AFFINITY BIOTINYLATION - NONRADIOACTIVE METHOD FOR SPECIFIC SELECTIONAND LABELING OF CELLULAR PROTEINS

Biotinylation has become a popular alternative to radioiodination for labeling cell surface proteins, whereas labeling of the total cellular protein pool is usually achieved metabolically with [S-35]methionine and [S-35]cysteine. In this paper we describe a new technique in which total cellular lysate proteins that have been affinity bound to a sol id phase are labeled efficiently with biotin. This labeling technique is preferable to direct biotinylation of cell lysate since the unreact ed biotin can be readily removed from the sample by washing. The affin ity step permits preselection of the molecules to be labeled, thereby decreasing the potential for nonspecific binding during subsequent imm unoprecipitation. We applied this affinity biotinylation method to a h uman cellular lysate in order to preselect the total glycoprotein pool for subsequent immunoprecipitation of HLA class I, Following immunopr ecipitation, SDS-PAGE, and Western blot, the biotinylated protein coul d be readily revealed by enhanced chemiluminescence. The results were comparable to those obtained by radiometabolic labeling and Western bl ot using a monoclonal antibody probe. Overall, the affinity biotinylat ion method is faster and more practical than conventional radiolabelin g, without any loss insensitivity. (C) 1997 Academic Press.