Rabbit anti human histone h3 mab d1h2

Livers were homogenized in extraction buffer pH 7.5, 50mM HEPES, 2mM EGTA, 2mM EDTA, 130mM NaCl, 10mM NaF, 20mM β-glycerophosphate, 2mM sodium pyrophosphate, 1mM sodium vanadate, 0.5mM PMSF, 0,1% nonidet P-40, 2mM benzamidine, 1mM TLCK, 10μg/ml leupeptin, 10% glycerol, with protease inhibitors (Complete, Roche, Indianapolis, IN), followed by sonication for 10 sec. Protein concentration was determined using Bradford reagent (Bio-Rad Laboratories, Hercules, CA). 100μg of denatured proteins were separated by SDS-PAGE (Mini-PROTEAN TGX and Criterion TGX Gels 10%, Bio-Rad Laboratories) and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat, dry milk in Tris-buffered saline with 0.05% Tween-20 for 1h at 25C, then incubated with primary antibodies overnight at 4C [Rabbit anti-human PPARγ mAb (C26H12), 1/500; Rabbit anti-human Histone H3 mAb (D1H2), 1/1000 (Cell Signaling Technology)], washed and incubated with secondary antibodies for 2h at 25C [Goat Anti-Rabbit IgG (H+L)-HRP Conjugate, 1/2000 (Bio-Rad Laboratories)]. After washing, SuperSignal® WestFemto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL) was added and the light signal detected and analyzed using a C-Digit Blot Scanner and Image Studio Lite Ver 3.1 (Li-Cor Biosciences, Lincoln, NE).