Quantitative Western Blot Analysis of αII-Spectrin

EDL muscles were secured to approximate in situ length and incubated in either standard PSS or 1.2% BaCl₂ in PSS for 1 h at 37 °C then frozen in liquid nitrogen. Following homogenization, protein concentration of the supernatant was quantified with the Bradford method (Cat. # 5000006; Sigma). Protein concentration of each sample was normalized in 4x Laemmli sample buffer (Cat. # 1610747, Bio-Rad; Hercules, CA, USA) containing 5% dithiothreitol. Samples were loaded on 4–20% gradient Mini-Protein TGX gels (Bio-Rad) for electrophoresis and transferred to LF-PVDF membranes (Millipore; Burlington, MA). Following 2 h blocking in 5% milk, membranes were incubated overnight at 4 °C and again for 3 h at 25 °C in primary antibody raised against αII-spectrin (1:250, Cat. # sc48382, Santa Cruz; Dallas, TX, USA). A secondary antibody (Alexa Fluor 800 IgG, 1:5000; Cat. # 926–32,212, Li-Cor Biosciences; Lincoln, NE, USA) was used to quantify protein differences with an Li Cor Odyssey Fc imaging system. Western blots were normalized to total protein according to the recommendations for fluorescent Western blotting [22 (link)] using Revert total protein stain (Cat. # 926-11010, Li-Cor). The 40 kDa bands correlate with the total protein in each lane and are shown to represent equal protein loading [23 (link), 24 (link)].