Simple Antibody Biotinylation
The HOOK™ IgG Biotinylation (Amine) kit is designed for the efficient biotinylation of IgG molecules by immobilizing the IgG molecules on a solid support (see 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 immobilized IgG molecule is then incubated with NHS-dPEG4-Biotin solution to biotinylated the free amine groups. The free NHS-dPEG4-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 3-5 biotin molecules per IgG molecule.
The HOOK™ IgG Biotinylation (Amine) kit offers an advantage over stand ard 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 NHS-dPEG4-Biotin (See Appendix A for structure and coupling reaction). The most widely used amine reactive biotinylation reagents are the water insoluble N-hydroxysuccinimide (NHS) esters or the water soluble N-hydroxysulfosuccinimide (sulfo-NHS) esters. The addition of a charged sulfonate (SO3-) on the N-hydroxysuccinimide ring of the sulfo-NHS esters results in their solubility in water (~10mM), but not permeable to plasma membranes. The solubility and impermeability to plasma membranes makes them ideal for studying cell surface proteins as they will only react with the protein molecules on the outer surface of plasma membranes. The reactions of the NHS and sulfo-NHS esters with amines are virtually identical leading to the formation of an amide bond and release of NHS or sulfo-NHS.
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 NHS-dPEG4-Biotin have a reduced occurrence of aggregation compared to non-PEG biotinylation reactions. HOOK™ IgG Biotinylation (Amine) kit is designed for the coupling of 1-10mg protein in 1ml buffer, suitable for 8 couplings.
Also available: HOOK™ IgG Biotinylation (Sulfhydryl) for the partial reduction and biotinylation of free sulfhydryl residues.
Features
- 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
Applications
- For the efficient and simple labeling of antibodies with biotin