Agarose coupled protein a g beads

2 × 10⁶ HeLa cells were seeded in 100 mm-cell culture plates and V5-tagged plasmids were transfected using PEI (as described in Cell culture and transient transfection) on the following day. 48 h post transfection, cells were lysed in lysis buffer (250 mM NaCl, 50 mM Tris-HCl pH 7.5, 10% glycerol, 1% Triton X-100 with protease inhibitor cocktail) and V5-tagged proteins were immunoprecipitated using V5 antibody and immobilized to agarose-coupled protein A/G beads (Roche, cat. nos. 11-134-515-001 and 11-243-233-001) overnight. Protein bound beads were washed with the lysis buffer and used for in vitro kinase assay. Kinase assay was performed using dephosphorylated myelin basic protein (MBP) (13-110, Merck) as a substrate in 25 mM Tris (pH 7.5), 5 mM β-glycerolphosphate, 2 mM DTT, 0.1 mM Na₃VO₄, 10 mM MgCl₂ and 5 mM ATP (Enzo, BML-EW9805-0100) in a final volume of 40 μL. The kinase assay mixture was incubated with the immunoprecipitated V5-tagged proteins for 30 min at 30 °C. The kinase reaction was terminated by adding 20 μL SDS-sample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 10 mM DTT and bromophenol blue) followed by boiling at 95 °C for 10 min. The samples were loaded onto 12% SDS–PAGE gels and subjected to immunoblotting analysis.