Cell Surface Biotinylation Isolation

Cell-surface biotinylation was carried out as previously described [5 (link)] using the Pierce cell-surface protein isolation kit (Thermo Scientific) as per the manufacturer’s instructions. Briefly, cells were labelled with sulfosuccinimidyl-2-[biotinamido] ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin) in PBS (Mg/Ca) for 30 min on ice, the unreacted biotin was quenched with 100 mM glycine and protein content in cells assayed by BCA after lysis. To precipitate biotinylated surface proteins, equal protein amounts from cell lysates were incubated with High Capacity Neutravidin Agarose (Thermo Scientific). Non-bound ‘intracellular’ proteins were separated before agarose was washed with ice-cold ‘high salt’ wash buffer (500 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.5) and ice-cold ‘no salt’ wash buffer (10 mM Tris-HCl pH 7.5). Bound proteins were eluted with SDS-PAGE sample buffer containing 50 mM DTT, before analysis by western blotting.