EGLN3/PHD3 Antibody - BSA Free

Novus Biologicals | Catalog # NB100-303

Novus Biologicals
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Key Product Details

Species Reactivity

Validated:

Human, Mouse, Rat, Primate

Cited:

Human, Mouse, Rat, Primate

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Immunoblotting, Immunocytochemistry/ Immunofluorescence, Simple Western, Immunoprecipitation, Chromatin Immunoprecipitation, Chromatin Immunoprecipitation (ChIP)

Cited:

Immunohistochemistry-Paraffin, Western Blot, Immunoblotting, Immunocytochemistry/ Immunofluorescence, Immunoprecipitation, Chemotaxis

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all EGLN3/PHD3 Primary Antibodies »

Product Specifications

Immunogen

A synthetic peptide made to the C-terminus of humanPHD3/HIF Prolyl Hydroxylase 3. [LocusLink ID 112399]

Reactivity Notes

Primate reactivity reported in scientific literature (PMID: 23732909). Rat reactivity reported in scientific literature (PMID: 23545307).

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

27 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for EGLN3/PHD3 Antibody - BSA Free

Western Blot: EGLN3/PHD3 AntibodyBSA Free [NB100-303]

Western Blot: EGLN3/PHD3 AntibodyBSA Free [NB100-303]

Western Blot: EGLN3/PHD3 Antibody [NB100-303] - Whole cell protein from MEF cells was separated on a 12% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 2 ug/mL anti-EGLN3/PHD3 in 1% milk, and detected with an anti-rabbit HRP secondary antibody using chemiluminescence.
Simple Western: EGLN3/PHD3 AntibodyBSA Free [NB100-303]

Simple Western: EGLN3/PHD3 AntibodyBSA Free [NB100-303]

Simple Western: EGLN3/PHD3 Antibody [NB100-303] - Image shows a specific band for PHD3/HIF Prolyl Hydroxylase 3 in 0.5 mg/mL of Hypoxic HeLa cell lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.
Immunohistochemistry-Paraffin: EGLN3/PHD3 Antibody - BSA Free [NB100-303]

Immunohistochemistry-Paraffin: EGLN3/PHD3 Antibody - BSA Free [NB100-303]

Immunohistochemistry-Paraffin: EGLN3/PHD3 Antibody [NB100-303] - LDL Receptor was detected in immersion fixed paraffin-embedded sections of human liver cancer using rabbit anti-human antibody (NB100-303) at 1:3000 overnight at 4C. Tissue was stained using the VisuCyte anti-rabbit HRP polymer detection reagent (VC003) with DAB chromogen (brown) and counterstained with hematoxylin (blue).
Western Blot: EGLN3/PHD3 AntibodyBSA Free [NB100-303]

Western Blot: EGLN3/PHD3 AntibodyBSA Free [NB100-303]

EGLN3-PHD3-Antibody-Western-Blot-NB100-303-img0010.jpg
Immunocytochemistry/ Immunofluorescence: EGLN3/PHD3 Antibody - BSA Free [NB100-303]

Immunocytochemistry/ Immunofluorescence: EGLN3/PHD3 Antibody - BSA Free [NB100-303]

Immunocytochemistry/Immunofluorescence: EGLN3/PHD3 Antibody [NB100-303] - HeLa cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton X-100. The cells were incubated with anti-HIF Prolyl Hydroxylase 3 (NB100-3030) at 1:200 overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at 1:500. Alpha tubulin, DM1A (NB100-690) was used as a co-stain at 1:1000 and detected with an anti-mouse DyLight 550 (Red) at 1:500. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Immunohistochemistry: EGLN3/PHD3 Antibody - BSA Free [NB100-303]

Immunohistochemistry: EGLN3/PHD3 Antibody - BSA Free [NB100-303]

Immunohistochemistry: EGLN3/PHD3 Antibody [NB100-303] - PHD3 was detected in immersion fixed paraffin-embedded sections of human heart using Rabbit Anti-Human EGLN3 polyclonal Antibody (NB100-303) at 5 ug/mL for 1 hour at room temperature followed by incubation with the Anti-Rabbit IgG VisUCyte(TM) HRP Polymer Antibody (VC003). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to nuclei.
Immunocytochemistry/ Immunofluorescence: EGLN3/PHD3 Antibody - BSA Free [NB100-303]

Immunocytochemistry/ Immunofluorescence: EGLN3/PHD3 Antibody - BSA Free [NB100-303]

Immunocytochemistry/Immunofluorescence: EGLN3/PHD3 Antibody [NB100-303] - Analysis of PHD3 in ARPE-19 cells using anti-PHD3 antibody. Image from verified customer review.

Applications for EGLN3/PHD3 Antibody - BSA Free

Application
Recommended Usage

Chromatin Immunoprecipitation

reported in scientific literature (PMID 21620138)

Immunocytochemistry/ Immunofluorescence

reported in scientific literature (PMID 20801873)

Immunohistochemistry-Paraffin

reported in scientific literature (PMID 25161887)

Immunoprecipitation

1:10-1:500. Use reported in scientific literature (PMID 15721254)

Simple Western

1:1000

Western Blot

1:500-1:2000
Application Notes
In Western blot, a band is seen at approx. 27 kDa. This antibody has been tested against HeLa and MEF cell lysates. In Simple Western only 10 - 15 uL of the recommended dilution is used per data point.
See Simple Western Antibody Database for Simple Western validation: Tested in Hypoxic HeLa lysate 0.5 mg/mL, separated by Size, antibody dilution of 1:1000, apparent MW was 31 kDa. Separated by Size-Wes, Sally Sue/Peggy Sue.

Reviewed Applications

Read 1 review rated 5 using NB100-303 in the following applications:

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: EGLN3/PHD3

HIF prolyl 4-hydroxylases (PHDs) are proyl hydroxylase domain-containing enzymes (PHD1/ Egln2, PHD2/ Egln1, PHD3/ Egln3, and P4H-TM) which are known for their role in mediating physiological responses to hypoxic stress via modulation of HIF1alpha expression levels. HIF-alpha subunit is regulated by hydroxylation, both by a family of PHDs leading to ubiquitination and proteasomal degradation, and by transcriptional inactivation following asparaginyl hydroxylation by FIH (factor inhibiting HIF). When oxygen levels are normal, HIF Prolyl Hydroxylase 3 (HIF-PH3/ PHD3/ Egln3) hydroxylates a specific proline found in HIF1A'a NODD/CODD domains and also hydroxylates HIF2A with preference for CODD site for HIF1A/HIF2A. Once hydroxylated, HIFs undergo proteasomal degradation via von Hippel-Lindau (VHL) ubiquitination complex. Upon hypoxic trigger, the hydroxylation reaction is tempered which let HIFs to escape degradation process leading to their nuclear translocation, heterodimerization with HIF1B, and increased expression of hypoxy-inducible genes. HIF-PH3 is the most important isozyme in limiting physiological activation of HIFs (particularly HIF2A) in hypoxia. Besides HIFs, in hypoxic conditions HIF-PH3 also hydroxylates PKM (thereby limiting glycolysis) and hydroxylates/regulates the stability of ADRB2. In cardiomyocytes, HIF-PH3 inhibits Bcl2's anti-apoptotic effect by disrupting the BAX-BCL2 complex, and in neurons, HIF-PH3 has a NGF-induced proapoptotic effect mediated via CASP3 activity regulation. HIF-PH3 also plays a crucial role in DNA damage response by hydroxylating TELO2, promoting its interaction with ATR which is required for activation of the ATR/CHK1/p53 pathway.

Long Name

Egl Nine Homolog 3/Prolyl Hydroxylase Domain-containing Protein 3

Alternate Names

HIFPH3, PHD3

Gene Symbol

EGLN3

Additional EGLN3/PHD3 Products

Product Documents for EGLN3/PHD3 Antibody - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

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Product Specific Notices for EGLN3/PHD3 Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Citations for EGLN3/PHD3 Antibody - BSA Free

Customer Reviews for EGLN3/PHD3 Antibody - BSA Free (1)

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Customer Images

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  • Name: Helder Andre
    Application: Immunofluorescence
    Sample Tested: ARPE-19 cells
    Species: Human
    Verified Customer | Posted 05/14/2015
    ARPE-19 cells
    EGLN3/PHD3 Antibody - BSA Free NB100-303

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Protocols

View specific protocols for EGLN3/PHD3 Antibody - BSA Free (NB100-303):

Western Blot Protocol
1. Perform SDS-PAGE on samples to be analyzed, loading 35 ug of sample lysate per lane.
2. Transfer proteins to membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain according to standard Ponceau S procedure (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot.
5. Block the membrane using standard blocking buffer for at least 1 hour.
6. Wash the membrane in wash buffer three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate 1 hour at room temperature.
8. Wash the membrane in wash buffer three times for 10 minutes each.
9. Apply the diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions.
Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%.

Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

FAQs for EGLN3/PHD3 Antibody - BSA Free

Showing  1 - 3 of 3 FAQs Showing All

  • Q: Dear sirs, which anti-PHD3 antibody would you recommend for western blot of murine tissues? I have used the NB100-303 and I am able to detect the protein in cells, but not in heart samples. could you also recommend a protocol with Ab dilutions, solutions, transfer, etc?

    A: In regards to your inquiry I am sorry to hear you have not had success with heart samples on NB100-303. We have another PHD3 antibody, NBP1-30328, that may be useful to you. It is guaranteed for mouse samples and has a sample size available if you wished to purchase and test first at a lower cost to you prior to committing to it.

  • Q: Do you have any information on possible antibody dilution to use for ChIP applications?

    A: We generally use around 5 x106 mammalian cells with 1 - 2 ug of NB100-303 (EGLN3/PHD3 Antibody) Therefore the amount used in ChIP with this antibody is consistent with our ChIP general protocol. 

  • Q: We are currently using the above PhD3 antibody and it is working well. However we are hoping to generate different truncation mutants of PhD3 and was wondering if the antibody will recognise these mutants. The data sheet indicates that the antibody was raised against the C-terminus of PhD3. Would it be possible to indicate where in the C-terminus the peptide is located. If the exact sequence is commercially sensitive perhaps you could tell us if the peptide is located in the fragment of 1-177 since we would like to make this mutant but are unsure if the antibody will recognise the fragment.

    A: The immunogen region is located in C-terminus. Specifically the immunogen is located within the range of amino acid 177 to the C-terminus. If your truncated protein only contains amino acids 1-177, this antibody will NOT recognize this protein.

  • Q: Dear sirs, which anti-PHD3 antibody would you recommend for western blot of murine tissues? I have used the NB100-303 and I am able to detect the protein in cells, but not in heart samples. could you also recommend a protocol with Ab dilutions, solutions, transfer, etc?

    A: In regards to your inquiry I am sorry to hear you have not had success with heart samples on NB100-303. We have another PHD3 antibody, NBP1-30328, that may be useful to you. It is guaranteed for mouse samples and has a sample size available if you wished to purchase and test first at a lower cost to you prior to committing to it.

  • Q: Do you have any information on possible antibody dilution to use for ChIP applications?

    A: We generally use around 5 x106 mammalian cells with 1 - 2 ug of NB100-303 (EGLN3/PHD3 Antibody) Therefore the amount used in ChIP with this antibody is consistent with our ChIP general protocol. 

  • Q: We are currently using the above PhD3 antibody and it is working well. However we are hoping to generate different truncation mutants of PhD3 and was wondering if the antibody will recognise these mutants. The data sheet indicates that the antibody was raised against the C-terminus of PhD3. Would it be possible to indicate where in the C-terminus the peptide is located. If the exact sequence is commercially sensitive perhaps you could tell us if the peptide is located in the fragment of 1-177 since we would like to make this mutant but are unsure if the antibody will recognise the fragment.

    A: The immunogen region is located in C-terminus. Specifically the immunogen is located within the range of amino acid 177 to the C-terminus. If your truncated protein only contains amino acids 1-177, this antibody will NOT recognize this protein.

  • Q: Dear sirs, which anti-PHD3 antibody would you recommend for western blot of murine tissues? I have used the NB100-303 and I am able to detect the protein in cells, but not in heart samples. could you also recommend a protocol with Ab dilutions, solutions, transfer, etc?

    A: In regards to your inquiry I am sorry to hear you have not had success with heart samples on NB100-303. We have another PHD3 antibody, NBP1-30328, that may be useful to you. It is guaranteed for mouse samples and has a sample size available if you wished to purchase and test first at a lower cost to you prior to committing to it.

  • Q: Do you have any information on possible antibody dilution to use for ChIP applications?

    A: We generally use around 5 x106 mammalian cells with 1 - 2 ug of NB100-303 (EGLN3/PHD3 Antibody) Therefore the amount used in ChIP with this antibody is consistent with our ChIP general protocol. 

  • Q: We are currently using the above PhD3 antibody and it is working well. However we are hoping to generate different truncation mutants of PhD3 and was wondering if the antibody will recognise these mutants. The data sheet indicates that the antibody was raised against the C-terminus of PhD3. Would it be possible to indicate where in the C-terminus the peptide is located. If the exact sequence is commercially sensitive perhaps you could tell us if the peptide is located in the fragment of 1-177 since we would like to make this mutant but are unsure if the antibody will recognise the fragment.

    A: The immunogen region is located in C-terminus. Specifically the immunogen is located within the range of amino acid 177 to the C-terminus. If your truncated protein only contains amino acids 1-177, this antibody will NOT recognize this protein.

Showing  1 - 3 of 3 FAQs Showing All
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Associated Pathways

HIF Enhancer Pathways HIF Enhancer Pathway Thumbnail