Ribonucleotide Reductase Inhibiting Protein SML1 抗体

Cell preparation for western blot: cells were harvested by centrifugation (4000 x g , 6 min, 4 °C). Supernatant was discarded and cells were resuspended in 500 µl cold TCA buffer (20 mM Tris, pH 8, 50 mM ammonium acetate, 2 mM EDTA, 1 tablet/10 ml of Complete Mini Protease inhibitor cocktail with EDTA (Roche Diagnostics GmbH)). 500 µl 0.5 mm Zirconia/Silica Beads (BioSpec Products, Inc. 11079105z) and 500 µl cold 20 % TCA was added. Samples were vigorously vortexed twice for 30 sec (kept on ice in between). 750 µl from the liquid phase was transferred into a fresh Eppendorf tube. Samples were centrifuged for 10 min (20000 x g, 4 °C). The pellet was resuspended in 300 µl TCA-Laemmli buffer and boiled for 10 min at 100 °C.