IC50: cell-surface protein

Sulfo-NHS-SS-biotin is a cell-surface-labeling reagent, it is a negatively charged reagent and does not permeate cell membranes. Sulfo-NHS-SS-biotin reacts with primary amines (NH2), such as lysine side-chains, or the amino-termini of polypeptides. Sulfo-NHS-SS-biotin has a sulfonate group and prevents it from permeating cell membranes. Thr group’s cleavable spacer arm enables initially biotinylated proteins to be released from streptavidin affinity columns. Sulfo-NHS-SS-biotin (1 mg/ml; 15 min) is applied in cells in monolayer culture (washed by ice-cold PBS). The biotinylation reactions are terminated with 100 mM glycine in PBS. After washing with PBS, cell extracts are prepared in RIPA buffer with protease inhibitor cocktail ( HY-K0010 ). Biotinylated membrane proteins are precipitated with streptavidin-sepharose and proteins are eluted with SDS sample buffer.