Mouse anti flag m2 magnetic beads

Cells were rinsed with ice-cold Dulbecco’s Phosphate-Bufferd Saline (DPBS) twice before harvested with Co-IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, and 1% Triton X-100) supplemented with Roche complete protease inhibitor cocktail. The lysates were then rotated for an hour at 4 °C and subject to centrifugation (16,000×g, 15 min, 4 °C) to remove cell debris and nucleus. The supernatant was collected as the total cellular protein. Protein concentrations were determined by the MicroBCA assay (Pierce). Western blot analysis was performed using appropriate antibodies. Band intensities were quantified using the Image J software from the NIH.
Cell lysates (500 μg) were precleared with 30 μL of protein A/G Plus-agarose beads (Santa Cruz Biotechnology) and 1.0 μg of normal mouse IgG for 1 hat 4°C to remove nonspecific binding proteins. The precleared cell lysates were incubated with 30 μL mouse anti-Flag M2 magnetic beads (M8823, Sigma Aldrich) or normal protein A/G plus agarose beads (negative control for nonspecific binding) overnight at 4 °C. The magnetic beads were collected using a magnetic separation stand (Promega) and washed three times with Co-IP buffer. Flag-tagged proteins were eluted by incubation with 30 μL of SDS loading buffer in the presence of β-mercaptoethanol. The immunopurified eluents were separated in an 8% tris-glycine gel, and Western blot analysis was performed.