You will want to remove any sodium azide (which interferes with the modification reaction) or gelatin (which has Lys). The azide can be removed by the desalting column or buffer exchange column included in the HRP kit. The gelatin is more of an issue because of its size – will not be removed by the buffer exchange (size exclusion column). Commonly used additives include preservatives such as sodium azide, thimerosal, protein stabilizers such as BSA or gelatin and/or small molecule additives such as glycine or trehalose. Protein carriers such as BSA or gelatin must be removed before labeling can proceed. Remove and purify away all protein carriers such as BSA or gelatin by affinity or other chromatographic methods, re-adjust the initial antibody concentration to 1 mg/mL. or, you can ask the antibody vendor to NOT add any stabilizers to your antibody preparation. The antibody-HRP conjugate should also be stored at 4ºC, and should be stable for at least one year.
The amines in a glycine or Tris-based buffer WILL interfere with optimal labeling of the antibody with HyNic, since HyNic reacts with amino groups. The All-in-One kit protocol buffer-exchanges your antibody into a phosphate-based buffer, free of amines, so the labeling reaction is optimized. The buffer exchange part of the protocol only takes a few minutes and is simple to perform.
All-in-One kits are the only kits that allow you to isolate virtually 100% pure antibody conjugate without HPLC/FPLC column purification. The All-in-One Kits used for antibody labeling are the only products of their kind that convert 100% of antibody to conjugate, making purification easy with the TriLinkmini-purification spin columns. All-in-One kits are the only kits for making high yielding, pure, high-quality antibody conjugates.
. Many of the biotin labeling kits from Pierce use an NHS-Biotin reagent WITHOUT the chromogenic fluorophore. Using a chromogenic biotin reagent offers MANY advantages…you can use simple UV quantitation to determine biotin incorporation – with no need for the HABA assay, an additional assay required to determine the extent of biotin incorporation. There are several other advantages as well, which are listed on our website. We offer a variety of formats of this reagent – both in kits and as standalone products. TriLink’s biotin product page provides an overview of our product offerings. If you perform a significant amount of biotin labeling, one of these other options may work well for you. Another consideration: we can provide you with a much higher level of technical support than can Pierce. As the inventor and developer of this reagent/technology, and having years of experience with custom conjugations and product development, we can better assist you with your projects and help you optimize biotin labeling for any of your applications. Also, we can offer discounts to you for volume purchases – either in kit format or in any custom packaging for your company.
Ascites fluid contains, not only your IgG antibody of interest, but also a number of other proteins found in the gut of the animal in which it is produced (in this case, mouse). An antibody in this form is not acceptable for conjugation because the biotinylation reaction targets amino acids on the antibody that would also be found in the other proteins. Your biotin/streptavidin assay would then be detecting, not only the activity of the IgG antibody, but also the activity of all the other proteins you have biotinylated. For biotinylation reactions we recommend only using purified antibodies. You could use our kit to biotinylate a secondary antibody to your Monoclonal Anti-Tyrosine Hydroxylase antibody which would detect antigen binding of the monoclonal. But if you biotinylate the ascites fluid, you will just have a nonspecific binding mess giving falsely high antigen binding results. Our biotinylation kit comes with desalting columns but is not equipped for specific purification of antibodies.
Our ChromaLink™ Biotin Labeling Reagent will work very well for your protein plasma serum biotinylation. The product that we recommend depends on how much you plan to biotinylate. Our ChromaLink™ Biotin Labeling Kit allows for 5 separate reactions of 1 mg of total protein each. This kit comes with desalting apparatus with a molecular weight cut off of 7,000 kDa. The ChromaLink™ labeling reagent labels any amino groups so these desalting columns will remove any amine-containing small molecules that may be in the serum (i.e., hormones, minerals, etc.). This will allow for labeling of large amine-containing biomolecules only. If you were hoping to biotinylate more than 5 mg of protein, then we would recommend bypassing the kit and just buying the ChromaLink™ reagent and then following the protocol on our website to biotinylate your proteins. It will be necessary for you to purchase the Zeba desalting columns from Thermo Scientific. We recommend that you make a few modifications to the protocol to account for the fact that you will be working with a variety of different plasma proteins. You should determine the protein concentration of your serum solution using a BCA assay (Thermo Scientific) or the A280 and then use an average protein mw (perhaps ~70,000 kDa) to determine the number of moles of protein in the solution. Then add 10 molar equivalents of our ChromaLink™ Biotin Labeling Reagent. All of the proteins should be sufficiently biotinylated with this method.
Our labeling reagent reacts with a primary amine group on any biomolecule. Therefore, in order to biotinylate your oligo, you would have to have it synthesized with an amino modification on either the 3′ or 5′ end. Most oligo companies can directly add a biotin onto the nucleotide as easily as they can add a primary amine group. The ChromaLink™ reagent, would therefore require an extra step and while it would be slightly more quantifiable – you could determine EXACTLY how many biotins/oligo- a mass spec from the oligo synthesis would provide similar information. In general, for biotinylation of proteins, peptides, or other amine-containing molecules, we recommend our product with confidence, but for oligos, we would just have the oligo synthesized with the biotin group.
While our ChromaLink™ reagent is certainly compatible with small proteins or biomolecules, our kit is comes with several desalting apparatuses that have a protein cut-off of 7,000 Da (7 kDa). We could certainly modify our kit for you and add some diafiltration units with a cut-off of 3 kDa. We would only recommend the diafiltration units if you have significant amount of your small protein, we are a bit concerned about the protein crashing out on the membrane. If you have an abundance of protein, we would certainly recommend trying the kit with this diafiltration procedure. Also the recommended amount of ChromaLink™ added to your small protein would, of course, have to be adjusted.
The gelatin will compete with the antibody with regards to biotin labeling. It is strongly recommended that the gelatin be removed prior to labeling. The kit will not work as well with the gelatin present. If the gelatin (or BSA) is not removed, a mixture of labeled antibodies and proteins results. There are antibody purification columns available from other vendors.
The ChromaLink™ biotin WILL bind to the metal resin. We have observed this before with some of our studies.
Once resuspended in buffer, you can store the biotinylated IgG control at 4oC and it should be good for 1–2 weeks.
It is unlikely that the protein is still on the column, as the resin excludes molecules with MW greater than 7,000. It is possible that you experienced some precipitation of your protein immediately after modification. If the protein concentration is too high (for some antibodies), aggregates can form and a precipitate forms. If this is the case, you might try lowering the Ab concentration to around 1 mg/mL. With some antibodies, lysine modification, removal of charged groups, can drastically affect solubility. If this is the case, you might try modifying with a lower MSR. Biotinylate your antibody again using a lower antibody concentration.
The bond that is formed between the antibody and the ChromaLink™ reagent is an amide bond, which is very stable in low pH. The ChromaLink™ chromaphore will not be affected by the low pH glycine buffer. Your protein G purification will not have any negative effect on the biotinylation of your protein.
Our reagent will biotinylate any amine-containing biomolecule. If your carbohydrate or lipid has been modified with amines, this kit will work for you. There are some disclaimers though.The kit (Cat. No. B-9007) comes with desalting apparatuses with a molecular weight cut off of 7,000 kDa. Therefore, if your biomolecules are smaller, you may be better off buying our reagent (Cat. No. B-1007) by itself and using a different desalting apparatus (diafiltration spin column or cassettes with smaller molecular weight cutoffs will work well). Also, while carbohydrates may be desalted with the Zeba desalting columns we provide, the hydrophobic nature of your lipid may lead to losses when desalted with these columns. The chromaphore will still absorb strongly at 354 nm, but our kit helps you determine the MSR (molar substation ratio) based on the concentration of the protein (usually determined by A280). You could assume 100% recovery of your biomolecule during the biotinylation reaction in order to determine the amount of biotinylation, but it won’t be as accurate as with protein using the A280 absorption. If you have a better way of determining the concentration of your carbohydrate or lipid, then you would be able to more accurately determine the biomolecule:biotin ratio.
Remember, only double stranded DNA that has biotin on one of the strands can be denatured. Typically, it will be a PCR product that was amplified with one biotinylated primer and the other must be non-biotinylated. If both strands are biotinylated you cannot easily remove the DNA from the beads without destroying the streptavidin completely.
1. After immobilization of biotinylated PCR DNA, place the 1.5 mL tube on a magnetic rack for 2 min to collect the beads onto the side of the tube.
2. Using a pipette, carefully remove and discard the clarified supernatant taking care not to dislodge the pellet.
3. To denature the non-biotinylated strand, add 200 µl of 100 mM NaOH (freshly prepared from 10N stock) to the beads and vortex to resuspend the beads. Incubate for 60 sec to denature the non-biotinylated strand.
4. Place the microfuge tube back on the magnetic rack for 2 min.
5. Carefully remove and discard the clarified supernatant.
6. Neutralize any residual base by washing the beads 2X with 500 µL of 0.1 M Tris-HCL, 150 mM NaCl.
7. The single stranded DNA immobilized on the beads is now ready for hybridization by addition of denatured complementary target DNA in a suitable hybridization buffer.
Therefore, if you were to use the biotinylated oligo with our streptavidin-coated magnetic beads, you would easily be able to capture the complementary strand of the biotinylated oligo and then remove it with 100 mM NaOH, which would quickly break the hydrogen bonds, leaving the DNA backbone intact. This type of de-hybridization scheme is much easier to buffer exchange than the detergent method.
Biotinylation probably won’t affect binding to an antibody although there are certainly risks. If you are using a monoclonal antibody they might be more site specific, especially with the N terminus (which will likely be modified by our biotinylation reagent). Additionally, the larger your antigen, the less likely biotinylation will inhibit binding. Really the only way to know is to try it, we have a lot of experience labeling proteins and antibodies and this really is the best way to modify without changing the biomolecule too much. Other reagents will not be as controllable and use more harmful chemistries that could harm your protein. I would recommend using lower biotinylation levels (depending on the size of your antigen, I would recommend not adding more than 10 molar equivalents) and it should work out fine. This reagent specifically labels amine groups on Lysine residues. The beauty of our linking reagent is that there is no reduction or oxidation step that would alter the hydroxyl groups on Asp or Glu residues. The kit has been developed so that only 3–8 biotins are labeled onto the antibody. There are about 50 Lys residues on any given antibody, so if you are only modifying three of them, the probability that you are hitting the antigen binding site is low. While we cannot guarantee that the antigen binding site is not being labeled, rendering it inactive, we rarely see a case where labeling causes the antibody to become inactive.
The optimal biotin MSR depends on the MW of the protein. We suggest 2–3 biotins for proteins < 50 kDa, 3–4 for 60–100 kDa proteins, and 4–6 for antibodies. This has to do with the percentage of Lys residues present, and how many charges are removed (which can and will affect the protein coming out of solution).
- We typically recommend an antibody concentration of at least 1 mg/mL for the biotinylation reaction. If infeasible to concentrate your IgG four fold, then increase the molar equivalents to 30 so that you can have a final MSR between 5 and 8. If you have a method of concentrating your antibody (e.g., Vivaspin Diafiltration filters, Cat. No. S-4004-010), I would recommend that first, then adding 20 equivalents of the ChromaLink™ reagent.
While the presence of lysine groups on an antibody is paramount, with regards to chicken IgY – it is too heavily glycosylated to work as well as bovine IgG with our kits. Although IgY will be labeled or modified with biotin to some extent, the sugars can interfere with binding to streptavidin in downstream applications. So the glycosylation can be an issue when working with IgY antibodies (in terms of downstream applications, where attachment to streptavidin is involved).
Some information below is borrowed from some other vendors, as this is a commonly asked question. If you do need to remove the captured antigen from the SA beads, you might also consider using a cleavable biotinylation reagent. Without a cleavable reagent, below are some of the common strategies used, which are harsh, yet necessary, to break the very strong bond.
The streptavidin-biotin interaction is the strongest known non-covalent, biological interaction between a protein and molecule. The bond formation between biotin and avidin is very rapid and, once formed, is unaffected by wide extremes of pH, temperature, organic solvents and other denaturing agents. Unless derivative forms of biotin or modified streptavidin have been adopted for your experiment, requiring a specific form and normally gentle way to dissociate biotin from streptavidin, often very harsh methods are required to dissociate the biotin from streptavidin which will denature the streptavidin. A couple of these methods are discussed below. For biotinylated proteins, boil the beads in 0.1% SDS or SDS-PHAGE buffer for 3 min.
One thing to try is to reduce the antibody concentration to around 1 mg/mL and use 20 equivalents of ChromaLink™ Biotin. This should help improve or increase the MSR. If a precipitate is observed, you may be experiencing another situation. Some antibodies are very sensitive to lysine modification—this could be one of them. Charges are being removed when lysine residues are modified, thereby affecting solubility of the protein.
The diluted biotin reagent should NOT be used after 1 day, even if frozen. Should be used fresh, as hydrolysis can occur. The sulfo-ChromaLink™ Biotin reagent is stable for an hour or for the day (at most) if kept at 4oC…but not longer than that. Hydrolysis occurs.
One should purchase reagents separately to save money. You can purchase 5 mL Zeba columns from Pierce. The 10X Modification Buffer (without azide), is S-4000-005. This is 5 x 1.5 mL for $50. You can get the ChromaLink™ reagent in bulk (5 x 1.0 mg) if preferred, which is B-1007-105 (for the water soluble version) or (10 mg), which is Cat. No. B-1007-010. The best modification conditions:
1. Protein concentration is 5–6 mg/mL.
2. Normally 6X equivalence of linker used, MSR will be 4–4.5. If you want high MSR, just increase to 8X or 10X.
3. The 5 mL Zeba desalt spin column is good for 500–2,000 µL.
4. Use 2.5 mL 1X MB or 1X PBS to pre-equilibrate column at 1,000 x g for 2 min (three times) before desalt (desalt at 1,000 x g for 3 min). Use 15 mL tube as collection tube.
There is only a minor pH difference between these 2 buffers.
10X Modification Buffer
1.0 M Sodium Phosphate
1.5 M Sodium Chloride
pH 8.0 @1X concentration
1.0 M Sodium Phosphate
1.5 M Sodium Chloride
pH 7.2 @ 1X concentration
If you are using the One-Shot kit or the standard ChromaLink™ kit, this is done in step 6 of the procedure (One-Shot protocol) or the second buffer-exchange step (standard kit) using the Zeba™ spin columns included in the kit. (2nd Buffer Exchange: remove excess labeling reagent)
For maximum sensitivity and reproducibility, it is important to avoid over-labeling, which leads to reduced immunoreactivity, or under-labeling, which leads to a weak signal. ChromaLink™ Biotin and DIG Labeling Kits are the only kits which enable quantification of the incorporated label with a simple UV scan of the labeled protein. No more HABA assays. By simply measuring the biotin- or DIG-labeled protein at 280 and 354 nm, and inserting the values into an easy-to-use calculator provided with the kit, you can determine the exact number of biotins or DIG on your antibody (MSR). Consistent, quantifiable labeling provides you with consistent, reliable, and reproducible results in your assays.