Simple Antibody Biotinylation
The HOOK™ IgG Biotinylation (Sulfhydryl) kit is designed for the efficient biotinylation of IgG molecules by immobilizing the IgG molecules on a solid support (Figure 1).
IgG molecules contain a histidine rich region in their Fc region and this feature is used to first immobilize the IgG molecules on a Nickel Chelating Resin support. The disulfide bonds of the immobilized IgG molecule are then reduced with the supplied Protein-S-S-Reductant™, with excess reductant being simply washed away. The reduced immobilized IgG molecule is then incubated with PEG2-Iodoacetyl-Biotin solution to biotinylated the free sulfhydryl groups. The free PEG2-Iodoacetyl-Biotin is washed away and the biotinylated IgG is eluted with an imidazole containing buffer. The biotinylation of the IgG on the Nickel Chelating resin results in the addition of 2-4 biotin molecules per IgG molecule.
The HOOK™ IgG Biotinylation (Sulfhydryl) kit offers an advantage over standard biotinylation reactions as the immobilization to the Nickel Chelating resin allows for the rapid removal of uncoupled biotin and therefore eliminates the need for further dialysis or desalting of the biotinylated antibody.
Biotin, a 244 Dalton molecule, exhibits an extraordinary binding affinity for avidin and streptavidin (Ka=1015 M-1). The biotinylated molecules are efficiently probed with avidin or streptavidin conjugated to reporter molecules, such as peroxidases or phosphatases. The use of biotin labeled proteins in ELISA, Western blotting and dot blotting is a popular technique.
The kit is supplied with a Nickel Chelating column that can be regenerated up to 10 times and our single-use aliquots of PEG2-Iodoacetyl-Biotin. PEG2-Iodoacetyl-Biotin is a sulfhydryl reactive biotinylation reagent that react with thiol groups at pH7.5-8.5 and forms stable thioether bonds. PEG2-Iodoacetyl-Biotin is water soluble, due to its polyethylene glycol (PEG) spacer arm. For specific reaction with sulfhydryls, limit the reaction to pH 7.5-8.5 and the molar ratio of iodoacetyl-biotin to protein such that the concentration of biotin is only slightly higher than the sulfhydryl concentration. Iodoacetyl reaction should be performed in dark to limit the formation of free iodine, which has the potential to react with tyrosine, tryptophan, and histidine residues.
The advantage of using a PEG (polyethylene glycol) biotinylation reagent is that the long hydrophilic spacer arm conveys its water solubility to the molecule that it is coupled to. This means that antibodies and other proteins labeled with our PEG2-Iodoacetyl-Biotin have a reduced occurrence of aggregation compared to non-PEG biotinylation reactions. HOOK™ IgG Biotinylation (Sulfhydryl) kit is designed for the coupling of 1-10mg protein in 1ml buffer, suitable for 8 couplings.
Also available: HOOK™ IgG Biotinylation (Amine) for the biotinylation of primary amines.
- Simpler antibody biotinylation
- Solid support technology eliminates dialysis/desalting antibody clean-up
- Suitable for 1-10mg antibody
- PEG Biotin reagent for reduced steric hinderance and increased labeled antibody solubility
- For the efficient and simple labeling of antibodies with biotin