||Whole cell lysate (denatured)
||Cell lysate was prepared by homogenization in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors. Cell debris was removed by centrifugation. Protein concentration was determined with Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The lysate was adjusted to 2.5 mg/ml, and then mixed with 5X Sample Buffer to become final 2 mg/ml in 1X Sample Buffer. The lysate was heated at 95°C for 5 min, and cooled rapidly.
||The lysate is ready to load on SDS-PAGE for Western blotting.
||In ready-to-use 1X Sample Buffer (50 mM Tris-HCl, 2% SDS, 10% glycerol, 300 mM 2-mercaptoethanol, 0.01% Bromophenol blue).