Quantifying Dystrophic Muscle Proteins

Quadriceps from wild type and dystrophic mice were homogenized in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1.0% IGEPAL, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with protease inhibitor cocktail (Complete, Roche) and phosphatase inhibitors (1 mM Na₃VO₄ + 1 mM NaF) using an UltraTurrex homogenizer followed by incubation on ice for 30 minutes and then cleared by centrifugation at 13,000 rpm for 10 min at 4 °C. Western blot was performed as previously described69 (link). The antibodies used were: rabbit anti-Serpinb1 (Sigma Prestige) at 1:500; rabbit anti-neutrophil elastase (Bioss) at 1:1000 and anti-rabbit HRP-conjugated secondary antibody (Santa Cruz Biotechnology) used at 1:10,000. HRP activity was visualized using the ECL Clarity^TM (BioRad) system and recorded on a LAS 4000 (GE) gel doc system.