Selective labeling of functional groups on membrane proteins or glycoproteins using reactive biotin derivatives and 125I-streptavidin

Abstract

Amino groups, sulfhydryl groups or oxidation-induced aldehydes on erythrocyte membrane proteins and/or glycoproteins, were reacted with biotinyl N-hydroxysuccinimide ester (BNHS), 3-( N-maleimido-propionyl) biocytin (MPB) or biocytin hydrazide (BCHZ), respectively. The detergent-lysed biotinylated samples were subjected to SDS-polyacrylamide gel electrophoresis, and the proteins were transferred onto nitrocellulose membranes. The blot was then incubated with a solution containing 125I-streptavidin, and processed for autoradiography. The advantages of this approach over previously reported procedures for labeling the three functional groups include the following: (a) extremely high sensitivity; (b) short exposure times of autoradiograms and relatively low levels of radioactivity; (c) singlestep radiolabeling procedures subsequent to processing and handling of gels and (d) no background labeling in control samples.