F0000000058
ID F0000000058
Title Supernatant
Subcellular Cytosol
Protein -
Peptide TiO2-HAMMOC
Note The cells were lysed with 20 mM HEPES (pH 7.5), 250 mM sucrose, 1.5 mM MgCl2, 10 mM KCl, and 0.5% Nonidet P-40, containing 1% protease inhibitor and left on ice for 5 min to obtain “supernatant fraction” for in vitro kinase reaction,. After sonication for 5 min, the lysate was centrifuged at 1500g for 10 min at 4 °C, and the supernatant was collected as “supernatant fraction”. To obtain the “pellet fraction” for in vitro kinase reaction, we followed the protocol of Dignam et al (Dignam, J. D. et al., Nucleic Acids Res. 1983, 11, 1475−1489.). In brief, the cells were lysed with lysis buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT) and left on ice for 10 min. After centrifugation at 1000g for 10 min at 4 °C, the cells were suspended in lysis buffer A and homogenized by a Dounce homogenizer. Then, the lysate was centrifuged at 1000g for 10 min at 4 °C. After removing the supernatant, the pellet fraction was centrifuged at 25 000g for 20 min at 4 °C again. The resultant pellet was solubilized with lysis buffer B (20 mM HEPES (pH 7.9), 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.5 mM DTT containing 1% protease inhibitor) and homogenized by the Dounce homogenizer. After stirring the solution for 10 min on ice, followed by centrifugation at 25 000g for 30 min at 4 °C, the supernatant was collected as “pellet fraction”. The buffer for both fractions was replaced with 40 mM Tris-HCl (pH 7.5) by ultrafiltration using an Amicon Ultra 10K at 14 000g and 4 °C. Protein amount was confirmed with a BCA protein assay kit and distributed to each of 100 μg aliquot.