Dystrophin antibody

Myoblasts were treated with CRISPR-Gold following the procedure described in S10. After differentiation, cells were harvested and protein extracts for Western blot analysis were made following the procedure of Lu QL, et al., 2005¹⁴ . Briefly, cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.5% deoxycholate, and 1% Nonidet P-40) containing proteinase inhibitor, and the total protein concentration was determined using the BCA protein assay kit (Thermo Scientific, USA). 150 µg of protein per sample was loaded onto a 4–20% gradient polyacrylamide gel (Bio-Rad, CA, Cat # 4561094), and run 6 hours at 35 volts, the protein content of the gel was then transferred onto nitrocellulose membranes. The membranes were blocked with 5% milk in Tris Buffered Saline (TBS) plus 0.1% Tween20 followed by an overnight incubation with dystrophin antibody (dilution 1 : 200, Abcam, UK) in TBS plus 0.1% Tween20 to detect dystrophin. GAPDH (dilution 1 : 2000, Thermo Scientific, USA) was used as a sample loading control. Blots were washed with 0.2% Tween-20 in TBS 3 times, and then incubated with a horseradish peroxidase (HRP)-coupled secondary antibody (Azures Biosystems, USA). Antibody binding was detected using an enhanced chemiluminescent detection system (Amersham).