Mini protean 3 unit

Trabecular muscle myosin heavy chain (MHC) composition was determined using SDS-PAGE gel electrophoresis on 4.5% and 7.5% acrylamide stacking and separating gels, respectively. Trabecular strips which were immediately flash frozen at harvest and stored at -80 ºC, were ground into a powder and dissolved in a solubilization buffer (62.5 mM Tris HCl, 10% glycerol, 2% SDS, 5% beta-mercaptoethanol, 0.02% bromophenol blue, pH=6.8) at a concentration of 1mg/40µl. Samples were boiled for three minutes, and immediately spun in a centrifuge at 5000 RPM for 15 minutes at 4 ºC. Solubilized samples were loaded (0.5 µl/well) into 0.75mm thick acrylamide gels and the gels were run in a Biorad Mini-Protean III unit for 10 hours at a constant voltage of 72V, and 25 hours at a constant current of 1mA/gel. Following electrophoresis, the gels were stained with Coomassie Blue for 60 minutes, then de-stained with a 50% ethanol, 7% acetic acid solution for 5 minutes, and a 5% ethanol, 7% acetic acid solution for at least 60 minutes. After de-staining, the gels were scanned with a Biorad scanner and analyzed with Image J for optical density (OD) to determine the relative composition of myosin heavy chains α (α-MHC) and β (β-MHC). Results will be expressed as the composition of α-MHC relative to total MHC (i.e. OD of α-MHC/(OD of α-MHC + OD of β-MHC)).

Myosin heavy-chain (MHC) and actin content was assessed using SDS-PAGE gel electrophoresis on 4.5% and 12% acrylamide stacking and separating gels, respectively. Myofibrillar protein extracts were dissolved in an SDS solubilization buffer (62.5 mM Tris HCl, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.02% bromophenol blue, pH 6.8) to a final concentration of 2.2 mg•μL -1 . Samples were boiled for 3 min and immediately spun at 5000 rpm for 15 min at 4°C. Solubilized samples were loaded (0.5 μL per well) into 0.75mm-thick acrylamide gels, and the gels were run in a Biorad Mini-Protean III unit at a constant voltage of 22 V at room temperature for 18 h. Following electrophoresis, the gels were stained with Coomassie Blue for 60 min, then destained with a 50% ethanol, 7% acetic acid solution for 5 min, and a 5% ethanol, 7% acetic acid solution for at least 60 min. After destaining, the gels were scanned with a Biorad scanner and analyzed with Image J to compare the optical density of MHC and actin between groups.

The MHC content was determined in each fiber after mechanical testing using SDS-PAGE gel electrophoresis on 4.5% and 7.5% acrylamide stacking and separating gels respectively (Toursel et al., 2000) . The gels were run in a Biorad Mini-Protean III unit at a constant voltage of 73V for 40 hours at 4°C, stained with Coomassie Blue and scanned. Following electrophoresis, fibers were divided into two groups: Type I fibers expressing MHC I and Type II fibers expressing MHC IId.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to analyze the protein patterns of gels, using a 5% stacking gel (pH 6.8) and a 10% running gel (pH 8.8) (Laemmli, 1970) . Insoluble protein gels (2 g) were combined with 10 mL of denaturant solution (100 mM Tris-HCl, pH 8.5) containing 2% SDS (wt/v) and 6 M urea. The mixture was homogenized and then incubated at 85 ºC for 30 min. Protein samples were then mixed at a ratio of 3:1 with the SDS-PAGE loading buffer (Beijing solarbio science & technology Co., Ltd., Beijing, China) containing SDS, β-mercaptoethanol, bromophenol blue, and glycerol. The samples
were kept in boiling water for 5 min. Then, the supernatants (5 µL) were placed into each well, and a mixture of the marker proteins was deposited in the first lane to identify polypeptides according to their apparent molecular weight (MW). Samples were subjected to electrophoresis at a constant current of 10 mA firstly, and then after samples entered the running gel, the current was changed to 25 mA using a Mini Protean III unit (Bio-Rad Laboratories, Inc., Richmond, CA, USA). Fixed proteins were stained with Coomassie Blue R-250 (0.125%, w/v) in 20% (v/v) ethanol, and destaining was performed by using 5% (v/v) acetic acid and 20% (v/v) ethanol.