HIF-1 alpha Antibody

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.

A:

NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.

Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequences

Lastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
Buffer: 50mM Sodium Borate
Preservative: 0.05% Sodium Azide

A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

A:

Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here

A: This particular antibody has not been tested in house, so unfortunately we do not have a specific protocol for this. We have a collaborator who tested this antibody in FACS and that image is on our datasheet. With the image is a brief of their protocol: 'Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - Hela cells were treated for 15 hrs with 200uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125ug anti- HIF-alpha and secondary FITC-conjugated goat anti-rabbit (in a 150ul reaction). Black- treated, anti-KLH control IgG; Red- untreated, anti-HIF1-alpha; Blue- treated, anti-HIF1-alpha.'

A:

CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A:

(1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
(5)    Last but not the least, Novus technical support team may be contacted via email

A: Most likely the 60kDa band is a degradation product of HIF1A in your samples. Since HIF-1 alpha degrades so rapidly in the presence of oxygen, our lab often sees bands at lower molecular weights. We have also experienced some non-specific banding around that molecular weight too in some tests.

A: You could try a few things to further inhibit the degradation.
1) Use the protease inhibitors (if you are not already using them).
2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

A:

A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

A:

HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

Please go through our hypoxia related FAQs, you should find them very informative.

Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

A:

Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.