Biotinylation of proteins in solution and on cell surfaces

This unit contains three protocols for biotinylation of isolated proteins: attaching biotin to primary amines (e.g., amino groups of lysyl residues); attaching biotin to sulfhydryls (i.e., thiol groups of cysteinyl residues); and attaching biotin to carbohydrate residues on proteins. As biotinylation of lysyl and cysteinyl residues may alter protein function, modification of protein carbohydrates, which is usually innocuous, may be preferable if intact protein function is required (e.g., for activity assays or affinity purification). Biotinylation of cysteinyl thiols requires that disulfide bonds in isolated proteins be reduced before labeling. Biotinylation of surface proteins on living cells is also described using mild reaction conditions. Finally, this unit includes a brief description of methods for detecting biotinylated proteins.