Immunoprecipitation of Flag Tagged Fusion Proteins

Harvested HEK293T cells were collected using 1 × KalB buffer supplemented with protein inhibitors as described above. Samples were placed on ice for 30 min, with pipetting up and down every 10 min, for complete cell lysis. Cell debris was pelleted at 13,000 rpm for 10 min at 4°C. Anti-Flag® M2 Magnetic Beads (Sigma-Aldrich) were washed 3 times, for 5 min each with 1 × Kal-B buffer before being loaded with cell lysates. Cell lysates were incubated with the beads with rotation at 4°C overnight. Flag-tagged proteins were eluted with 80 μl of 150 μg/ml Flag peptide (Sigma-Aldrich) with rotation at 4°C for 30 min. Eluted samples were processed for immunoblotting as described above.