Pierce streptavidin coated magnetic beads

biotin labeling in HFFs was initiated with the addition of 50 μM biotin (Sigma B4501; from a 100 mM stock dissolved in dimethyl sulfoxide [DMSO]) for 1 h and terminated by cooling cells to 0°C on wet ice and washing away excess biotin (3 to 5 washes with ice-cold PBS). Then, either the cells were fixed for subsequent staining for IFA, lysates were prepared for Western blot analysis (resuspended in cold RIPA and clarified with a 10,000 × g spin for 10 min at 4°C), or clarified lysates were subjected to enrichment of the biotinylated proteins by streptavidin bead enrichment. To enrich for biotinylated proteins, the lysates were incubated with prewashed Pierce streptavidin-coated magnetic beads (Thermo Fisher 88817), rotating overnight at 4°C. The beads were subsequently washed 2 times with 1 ml of RIPA lysis buffer, 1 time with 1 ml of 1 M KCl, 1 time with 1 ml of 0.1 M Na₂CO₃, 1 time with 1 ml of 2 M urea in 10 mM Tris-HCl (pH 8.0), and 3 times with 1 ml RIPA lysis buffer. With the exception of the samples submitted for LC-MS/MS analysis, for which a detailed protocol is included below, the biotinylated proteins were eluted from the beads by incubation with Laemmli sample buffer supplemented with BME (Bio-Rad) and 2 mM biotin at 90°C for 10 min.