Volume approximately of 1 cm3 of whole cell were take into a
1.5 mL microcentrifuge tube and let thaw on ice. Lysis buffer with phosphatase inhibitor cocktail (50 mM NaF, 100 mM β-glycerolphosphate, 20 mM sodium pyrophosphate)(Brown and Gordon, J. Biol. Chem. 259, 9480 (1984)) were mixed with sample. Cap the centrifuge tube and mix by manual homogenizer and vortexing vigorously for 20 minutes at 37 C. Centrifuge 20 minutes at 10000 rpm. At 4 C. Cautious decant supernantant of each sample into the new microcentrifuge tube.