Cell surface biotinylation

grown cells until 70-80% confluency in 10cm dish

place cells on ice and wash with ice-cold PBS

incubate cells for 30 min c on ice with PBS containing 2.5 mg/ml Sulfo-NHS-SS-biotin (Pierce).

stop the biotinylation reaction by washing 3 times for 5min with quenching solution (0.5% BSA and 100 mM glycine in PBS).

collect cells by scraping in PBS and centrifugation 500rpm,4°C

lyse cells by resuspending cell pellet RIPA buffer supplemented with protease inhibitor and incubating for 30min on ice

clear cell lysate by centrifugation (20min, 14000 gavg, 4°C) and collect supernatant

isolate biotinylated proteins with immobilized Neutravidin beads

wash neutravidin beads 2 times with RIPA buffer

add supernatant from step 7 to beads

incubate while rotating head-over-head 2h 0m 0s

wash neutravidin beads 3 times with RIPA buffer

Input and bound proteins were processed for Western blotting with 4x LDS loading buffer.

remove all RIPA buffer from neutravidin beads

add 4x LDS loading buffer

denature proteins for 10min, 70°C

proceed with Western blotting