Lightning-Link (R) Streptavidin Antibody Labeling Kit
Novus Biologicals | Catalog # 708-0010
Key Product Details
Kit Type
Conjugate
Product Summary for Lightning-Link (R) Streptavidin Antibody Labeling Kit
Our streptavidin antibody labeling kit enables the direct conjugation of streptavidin to any biomolecule with an available amine group. The researcher simply pipettes their antibody or other biomolecule into the vial of Lightning-Link Streptavidin and incubates for 3 hours.
| Features | Benefits |
| Quick and easy to use | Save time, no special knowledge required |
| No separation steps | 100% recovery - no antibody/protein loss |
| Can be used in a wide range of applications | Flexible |
| Freeze dried | Ships at ambient temperature, long shelf-life |
| Fully scalable (10 ug to 1 g or more) | Easy transfer from R&D to manufacturing |
| Stringently QC tested | Consistent high quality, excellent batch-to-batch reproducibility |
| Large number of labels available | Experimental flexibility |
| Reliable: nearly 300 references | Successfully used in many fields of research |
Streptavidin is a 53kDa protein purified from Streptomyces avidinii, which has widespread applications due to its very high affinity to the vitamin biotin. Streptavidin is a tetrameric molecule, composed of 4 13kDa monomers, each of which can bind a molecule of biotin.
The Lightning-Link Streptavidin kit is unique in the sense that it is the only DIY streptavidin labeling kit available on the market. Like our biotinylation kits, Lightning-Link Streptavidin has been optimised to create high quality conjugates and minimise assay background. There is also no need for a wash or desalting step. The way in which the kits are optimised means the antibody or protein of interest will be labeled with up to 3 streptavidin molecules each, maximising the opportunity for biotin to bind and create a biotin streptavidin complex.
Learn more about Lightning-Link™ Conjugation Kits by reading FAQs
For more information please check out these useful links!
Antibody Labeling Guide
Antibody Conjugation Illustrated Assay
Product Specifications
Application Notes
Kit Contents for Lightning-Link (R) Streptavidin Antibody Labeling Kit
- 1 or 3 glass vial(s) of Lightning-Link mix
- 1 vial of LL-Modifier reagent
- 1 vial of LL-Quencher reagent
Formulation, Preparation, and Storage
Concentration
Shipping
Storage
Background: Lightning-Link (R) Streptavidin
Additional Lightning-Link (R) Streptavidin Products
Product Documents for Lightning-Link (R) Streptavidin Antibody Labeling Kit
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Product Specific Notices for Lightning-Link (R) Streptavidin Antibody Labeling Kit
This product is manufactured by Abcam and distributed by Novus Biologicals.
This product is for research use only and is not approved for use in humans or in clinical diagnosis. This product is guaranteed for 1 year from date of receipt and this statement overrides any mentioned guarantee period on the limitations section of this products datasheet. Please contact technical@novusbio.com with questions.
This product is for research use only and is not approved for use in humans or in clinical diagnosis. Kits are guaranteed for 6 months from date of receipt.
Citations for Lightning-Link (R) Streptavidin Antibody Labeling Kit
Customer Reviews for Lightning-Link (R) Streptavidin Antibody Labeling Kit (2)
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Application: ELISASample Tested: RANTES proteinSpecies: HumanVerified Customer | Posted 05/03/2016
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FAQs for Lightning-Link (R) Streptavidin Antibody Labeling Kit
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
-
Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
-
Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
-
Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
-
Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
-
Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
-
Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
-
Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
-
Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
-
Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
-
Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
-
Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
-
Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
-
Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
-
Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
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Q: Can I label a peptide/protein/antibody other than IgG?
A: Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
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Q: Can non-antibody molecules be labeled?
A: Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.
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Q: Can you tell me if the Streptavidin in kit #708-0010 is wildtype, monomeric or monovalent? Basically I want to know how many biotin can bind to the streptavidin in this kit. Also, I am presuming that the each functionalized streptavidin in this kit can only link to one antibody. Is a one streptavidin to one antibody conjugation the correct assumption? Finally, do you have the molecular weight of the streptavidin in this kit in daltons?
A: Streptavidin is a 53kDa protein purified from Streptomyces avidinii, and is a tetrameric molecule, composed of four 13kDa monomers, each of which can bind a molecule of biotin. Therefore 4 biotins can bind to the streptavidin in this kit. A mixed population is always formed in protein:protein labeling. Some antibodies may get one label, others two etc. The average number can be influenced by the amount of antibody added. Unless you have just 1 reactive group on streptavidin it is impossible to guarantee that it cannot react with a second antibody molecule (We do not believe such a reagent exists). Our recommendations on how much antibody to add to the vial are based on assay performance in an internal test, and you should start there, but re-optimization for other applications could be carried out using the 10ug vials if required.
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Q: Do I need a wash or desalt step?
A: One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
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Q: Do I need to purify the conjugate?
A: No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.
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Q: Do my antibody and buffer fit the requirements?
A: Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.
Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page. -
Q: How does this kit conjugate antibodies to streptavidin? amine-thiol reaction or another one??
A: This kit works by labeling the free amine groups.
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Q: How many labels will bind to my biomolecule?
A: It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
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Q: How stable will my new conjugate be?
A: The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
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Q: I have just ordered 708-0010 and I would like to know if you have any literature available?
A: If you go to the Images, Reviews & Publications tab of the product datasheet you can view the publications available. Also, if you go to the Protocols & FAQs tab of the product datasheet you can view protocols for this product.
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Q: I need to know to which antibody domain or AA sequence the kit will bind SA or if there is a specific amino acid sequence that will be labeled, it would be helpful to know that as well
A: The Lightning Link chemistry binds Streptavidin to primary amine residues (primarily lysine) within the antibody sequence.
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Q: What are the best storage conditions for my new conjugate?
A: TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
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Q: What can I do if my antibody formulation does not fit the requirements?
A: If your antibody buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume,...). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
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Q: What functional groups do I need on my protein?
A: Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.
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Q: What is the difference between Lightning-Link® and Lightning-Link® Rapid?
A: The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.