Sml1, Suppressor of Mec1 lethality

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Product Description

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