Gradient tris hcl polyacrylamide gels for electrophoresis fractionation

Frozen NAc tissue was homogenized in 30 μl of RIPA buffer containing 10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X–100, 1% Sodium Deoxycholate, and protease inhibitors (Roche, Basel, Switzerland) using an ultrasonic processor (Cole Parmer, Vernon Hills, IL, USA). Protein concentrations were determined using a DC protein assay (Bio–Rad, Hercules, CA, USA), and 50 μg of protein were loaded onto 4–15% gradient Tris–HCl polyacrylamide gels for electrophoresis fractionation (Bio–Rad). Proteins were transferred to nitrocellulose membranes, blocked with Odyssey® blocking buffer (Li–Cor, NE, USA), and incubated overnight at 4°C with primary antibodies (BAZ1A: Bethyl A301–318A, 1/500; BAZ1B: Abcam ab51256, 1/500; SMARCA5: Abcam ab3749, 1/1000) in Odyssey® blocking buffer. After thorough washing with 1x Tris–Buffered Saline plus 0.1% Tween–20, membranes were incubated with IRDye® secondary antibodies (1/5000 to 1/10000; Li–Cor, Lincoln, NE, USA) dissolved in Odyssey® blocking buffer for 1 hr at room temperature. For analysis, the blots were imaged with the Odyssey® Infrared Imaging system (Li–Cor, Lincoln, NE, USA) and quantified by densitometry using ImageJ (NIH, Bethesda, Maryland, USA). The amount of protein blotted onto each lane was normalized to levels of GAPDH (Cell Signaling 2118, 1/30000).