Antiha sepharose

Cell lysates were prepared by resuspending the pellets from 150 to 200 OD₆₀₀ units in 800 μl lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EDTA pH 8, 0.5% NP-40, 1x complete EDTA-free protease inhibitor cocktail (Roche), 2 mM PMSF, 20 mM N-ethylmaleimide). To block phosphatase activity, 1x tablet of PhosSTOP, 10 mM NaF and 20 mM ß-glycerophosphate were added to the lysis buffer. Cells were lysed by bead beating (Precellys 24, Bertin instruments) with zirconia/silica beads (BioSpec Inc.) and lysates were cleared by centrifugation (800 g, 5 min). Clarified extracts were incubated with pre-equilibrated antiHA-sepharose (Roche), GFP-Trap (Chromotek) or IgG-agarose beads (Sigma) for 1.5 h at 4 °C, beads were washed four times with lysis buffer and two times with wash buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA pH 8). Proteins were eluted by boiling with 30 µl HU loading buffer and analyzed by immunoblotting.