Carboxyl groups, in the form of carboxy termini, aspartate residues or glutamate residues, can be targeted for biotin labeling using amine-derivatised biotin molecules. This reaction is mediated by a class of crosslinkers known as carbodiimides and results in the formation of an amide bond. The reaction with EDC, the most common carbodiimide crosslinker, is generally performed in an MES buffer at pH 4,5 to 5 and requires just minutes to complete.
Buffers containing primary amines (Tris, glycine, etc.) or carboxyls (acetate, citrate, etc.) must be avoided because they will quench the reaction. Phosphate buffers are also not recommended because they reduce the conjugation efficiency, although this effect can be overcome by adding more EDC.
Pentylamine-Biotin: Dissolves in aqueous solutions. Use the EDC crosslinker to couple this analogue to carboxyl (–COOH) groups. Coupling with EDC occurs at pH 4 to 6.
Amine-PEGn-Biotin: Water soluble, PEG-containing reagents with terminal primary amines (–NH₂). The PEG spacer arm is hydrophilic and confers greater solubility to labeled proteins compared to reagents having only hydrocarbon spacers. The amine group of these reagents can be reacted with carboxyl groups on carboxy termini, aspartate residues or glutamate residues using EDC.
Alkoxyamine-PEG-Biotin: Used for biotinylating macromolecules at carbohydrate groups that have been oxidised to form aldehydes. The alkoxyamine group reacts to carbonyls (aldehydes and ketones), resulting in an oxime linkage. Alkoxyamine-PEG-Biotin Reagents contain a multi-functional extended spacer arm that is a flexible, non immunogenic hydrophilic polyethylene glycol (PEG), which imparts water solubility to labeled molecules. Consequently, antibodies labeled with biotin alkoxyamine reagents exhibit less aggregation when stored in solution compared to antibodies labeled with reagents having only hydrocarbon spacers.
