β mercaptoethanol

1 million cells were lysed in 100μl of LBX in presence of cOmplete EDTA free Protease inhibitor cocktail (Roche). The lysates were sonicated for 20 minutes at 4°C in a Sonorex Digitec (model DT100) ultrasonic bath (Bandelin) and cleared by centrifugation at 16,000 g for 10 minutes at 4°C. The supernatant was stored at −20°C until blotting. 6X Laemmli buffer (12% SDS (v/v) (Euromedex), 58% Glycerol (v/v) (Pharmacia Biotech), 375mM Tris HCl pH 6.8, 30% β-mercaptoethanol (v/v) (Pharmacia Biotech), 0.0012% Bromophenol Blue Before (w/v) (Pharmacia Biotech)) was added to samples to a final concentration of 1X prior to gel run. Samples were boiled at 95°C for 20 minutes on a thermoblock, immediately chilled on ice and centrifuged at 16,000 g for 5 minutes. 15-40μl of samples were resolved on 4%–20% SDS-PAGE gels (Biorad) and transferred on nitrocellulose membrane (Biorad). Membranes were saturated and proteins were blotted with antibodies (listed in Key Resources Table) in 5% non-fat dry milk, PBS, 0.1% Tween buffer. ECL signal was recorded on a ChemiDoc Touch Biorad Imager. Data was analyzed with Image Lab (Biorad).