Pierce streptavidin ultralink resin

Transfected TSA 201 cells were rinsed twice with ice-cold PBS and then incubated with 0.2 mg/ml EZ-Link sulfo-NHS-LC-biotin (Thermo Fisher Scientific), freshly prepared in NES, for 1 h at 4 °C. Nonreactive biotin was quenched, and unbound biotin was removed by washing three times with stock buffer (25 mm Tris-HCl, 150 mm NaCl, 10 mm EDTA, pH 7.5). Cells were lysed in solubilization buffer (stock buffer, 1% Triton X-100, 1 mm PMSF, protease inhibitor cocktail (Roche Applied Science)) on a rotating wheel for 1 h at 4 °C and then centrifuged at 18,000 × g. A 50-μl aliquot of the supernatant was diluted in Laemmli sample buffer and stored as the “total” protein sample, whereas the remaining supernatant was added to 30 μl of washed Pierce® Streptavidin UltraLink® resin (Thermo Fisher Scientific) and incubated on a rotating wheel at 4 °C for 2 h. Resin was centrifuged at 10,000 × g and washed in stock buffer with 1% Triton four times, and then proteins bound to the resin were eluted by the addition of 50 μl of Laemmli sample buffer and stored as the “biotinylated” protein sample. Samples were then analyzed by SDS-PAGE and immunoblotting. Two wells of a 6-well plate of confluent cells were solubilized in 350 μl of solubilization buffer unless otherwise stated.

Biotin labeling was performed as previously described [43 ]. Cells were placed on ice and washed with phosphate buffered saline (PBS). EZ-link Sulfo-NHS-SS-Biotin (Thermo Scientific) was used to prepare biotinylation solution immediately before incubating cells for 30 min at 4°C. Cells were washed three times on ice and immediately lysed in lysis buffer (50 mM HEPES pH7.5, 137 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 2 mM EDTA, 2 mM Na₃VO₄, 10 mM NaF, 10% glycerol, protease inhibitor cocktail [Roche complete Mini, EDTA-Free]) for 30 min at 4°C. The cell lysate was centrifuged at 14,000 g for 30 min at 4°C. Whole cell lysates were used for detection by immunoblot and for immunoprecipitation with Pierce Streptavidin UltraLink Resin (Thermo Scientific) overnight at 4°C to form Avidin-Biotin complex (ABC). The ABC resin was washed in PBS three times, and precipitated proteins were analyzed by immunoblotting.