Santa cruz western blotting luminol reagents

The cell monolayers were washed with PBS and scraped in cell lysis buffer containing 50 mM Tris, 5 mM MgCl2·H20, 25 mM KCl, 2 mM EDTA, 40 mM sodium fluoride, 4 mM sodium orthovandate, 1% Triton X-100, and protease inhibitor cocktail (Roche). The cells were extracted on ice for 30 min including sonication, as necessary. The cell lysis solution was clarified (2000 rpm, 2 min), centrifuged (10,000 rpm, 10 min), and the supernatant was saved. Protein analysis of extract aliquots was performed using BCA protein assay kit (Pierce, Rockford, IL). Cell extracts (amounts equalized by protein concentration) were mixed with 2× Laemmli sample buffer and reducing agent and boiled for 5 min. Lysates were loaded on a 4–10% SDS polyacrylamide gel, and electrophoresis was carried out according to standard protocols. Proteins were transferred to a membrane (Trans-Blot Transfer Medium, Nitrocellulose Membrane; Bio-Rad Laboratories) overnight. The membrane was incubated for 2 h in blocking solution (5% dry milk in TBS-Tween 20 buffer). The membrane was incubated with appropriate primary antibody in blocking solution. After being washed in TBS-1% Tween buffer, the membrane was incubated in appropriate secondary antibody and developed using the Santa Cruz Western Blotting Luminol Reagents (Santa Cruz Biotechnology) on the Kodak BioMax MS film (Fisher Scientific).