Monitoring MHC-II Endocytosis and Recycling Using Cell-Surface Protein Biotinylation-Based Assays.

Most, if not all, plasma membrane proteins continuously undergo endocytosis and many rapidly recycle from endosomes back to the cell surface to maintain "stable" surface expression. We now describe a biochemical assay that is suited to follow the internalization and recycling kinetics of plasma membrane proteins. This assay involves biotinylation of plasma membrane proteins using sulfo-NHS-SS-biotin, a water-soluble, NHS-ester biotinylation reagent that contains a cleavable disulfide bond that allows for reversible labeling of proteins. Biotinylation is rapid and stable, and does not transfer from cell to cell, and the small size of the biotin probe does not affect cell function.