Cell Surface Biotinylation Sample Clauses

Cell Surface Biotinylation. HEK-293T/17 cells were transfected with 2µg of DNA (empty vector or receptor). At 24 h post-transfection, cells were placed on ice and washed with ice- cold PBS+Ca2+. Cells were then incubated with 10 mM Sulfo-NHS-Biotin (Thermo Scientific) in PBS+Ca2+ on ice for 1 h and then washed three more times with PBS+Ca2+ + 100 mM glycine to quench. Cells were harvested in 500 µl of lysis buffer (1% Triton X- 100, 10 mM Hepes, 50 mM NaCl, 5 mM EDTA, and protease inhibitor cocktail (Roche Diagnostics)) and lysed by end-over-end rotation for 30 min at 4°C. Cell debris was cleared by centrifugation, and soluble cell lysates were incubated with 50µl of streptavidin agarose beads (Thermo Scientific) for 1 h at 4°C. Beads were washed 3 times with lysis buffer and resuspended in 100 µl of Laemmli buffer. Biotinylated proteins were detected via Western blot, as described above.