||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 mg/ml.
||Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.
||Use it directly for immuno-precipitation, or heat lysate with SDS gel loading buffer to 95°C for 5 minutes followed by rapid cooling for western blot application. If dissociating conditions are required, add reducing agent prior to heating.
||In modified RIPA Lysis Buffer.