Stain free 4 20 precast gels

Muscle samples were homogenized in 10 volumes of lysis buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM egtazic acid, 1 mM EDTA, 100 mM NaF, 5 mM Na₃VO₄, 1% Triton X‐100, 1% sodium dodecyl sulfate (SDS), 40 mM β‐glycerophosphate, and protease inhibitor mixture (P8340; Sigma‐Aldrich)] and centrifuged at 10 000 g for 10 min (4°C). Sixty micrograms of protein extract was loaded into Stain‐Free 4–20% precast gels (4568095; Bio‐Rad) before electrophoretic transfer onto nitrocellulose membranes (Bio‐Rad; Trans‐Blot Turbo Blotting System). After transfer, the membranes were blocked with 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20 (Tris‐buffered saline‐T) containing 5% skimmed milk or BSA and incubated overnight at 4°C with primary antibodies. The membranes were then incubated for 1 h with a peroxidase‐conjugated secondary antibody. The immunoblots were revealed using a Pierce ECL kit (32106; Thermo Scientific), and proteins were visualized by enhanced chemiluminescence using the ChemiDoc Touch Imaging System and quantified with Image Lab™ Touch Software (version 5.2.1). Stain‐Free technology was used as the loading control. A large number of methodological studies have already validated this technology and explained its functioning in detail.61, 62, 63, 64, 65, 66, 67, 68

Gastrocnemius samples were homogenized in 10 volumes of lysis buffer and centrifuged at 10 000 g for 10 min (4°C). Sixty micrograms of protein extract were loaded into Stain‐Free 4–20% precast gels (4568095; Bio‐Rad) before electrophoretic migration and transfer onto nitrocellulose membranes (Bio‐Rad; Trans‐Blot Turbo Blotting System). Then, the membranes were blocked and incubated overnight at 4°C with primary antibodies. The membranes were then incubated for 1 h with a peroxidase‐conjugated secondary antibody (see Supporting Information for details).