Immunoprecipitation of Biotinylated Proteins

Isolation of biotinylated proteins was performed using streptavidin magnetic beads as previously described (⁴⁷). Streptavidin magnetic beads were washed twice with 1mL of RIPA lysis buffer. Whole cell lysate samples were incubated and rotated with 30μL of streptavidin magnetic beads and 500μL of RIPA buffer for 1 hour at room temperature. The beads were then collected using a magnetic rack, and the supernatant was removed and stored for later immunoblotting analysis. The remaining magnetic beads were washed twice with 1mL of RIPA lysis buffer, once with 1mL of 1M KCl, once with 1mL of 0.1M sodium carbonate, once with 1mL of 2M urea in 10mM Tris-HCl (pH 8.0), and then twice again with 1mL of RIPA lysis buffer. To elute biotinylated proteins from the magnetic beads, the samples were boiled for 10 minutes in 100μL of 25mM Tris, 50mM NaCl, 10mM DTT, 2% SDS, and 5mM Biotin. We then vortexed the beads briefly, and cooled the sample on ice. The samples were then placed on the magnetic rack and the eluate was collected and placed on ice.