Nupage mops sds buffer

In order to obtain total protein extracts, cells were lysed using either Laemmli lysis buffer (4% SDS, 20% glycerol, 120 mM Tris-HCl, pH 6.8) or RIPA buffer (150 mM NaCl, 1.0% IGEPALâ CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris-HCl, pH 8.0) supplemented with protease and phosphatase inhibitors and denatured for 10 min at 70 C. In order to prepare chromatin enriched protein fractions, cells were incubated with Pre-extraction Buffer (0.025 M HEPES-NaOH, pH 7.4, 0.05 M NaCl, 1 mM EDTA, 3 mM MgCl 2 , 0.3 M Sucrose, 0.5% Triton X-100) supplemented with protease inhibitors for 10 min on ice prior to lysis. Proteins were resolved by SDS-PAGE using NuPage 4-12% Bis-Tris 1 mm gel and NuPage MOPS SDS buffer (Invitrogen), at 200 V for 1 h. Proteins were transferred to PVDF membrane by semidry transfer for 1 h and 10 min at 65 mA. Membranes were blocked in 5% milk or BSA in TBS-0.1% Tween 20 for 30 min, and incubated with primary antibodies overnight at 4 C and secondary antibodies for 1 h at RT. Antibodies used are listed in the Key resources table. Primary antibodies were used at a dilution 1:1000 unless otherwise stated, while secondary antibodies were used at a dilution 1:5000. The signal was visualized with ECL substrate in an Amersham imager 600 (GE Healthcare).