The protein concentration of each TA muscle was optimized so that 0.4 μg of protein was added to each gel channel in duplicate. SDS-PAGE was performed on each concentrate with a 0.75 mm thick 6% acrylamide/30% glycerol separating gel (18 × 16 cm) and a 4% acrylamide/30% glycerol stacking gel. A silver staining kit (SilverQuest Silver Staining Kit, Life Technologies, New York, USA) was used to stain the gel for visualization of protein bands. Each silver-stained gel was imaged digitally. An investigator masked to rat age and treatment determined the optical density of each band using computer-assisted image analysis and densiometry (UN-Scan-IT gel Version 6.1, Silk Scientific, Inc, Utah, USA). The ratio of the density of an individual MHC isoform band to the total density within a column was used to determine the percentage of each MHC isoform per lane 19 ,44 (link),46 (link)-51 (link). Good to excellent reliability using these procedures has been demonstrated in previous studies 50 (link).
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Quantifying Muscle Fiber Type Composition
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Corresponding Organization : University of Wisconsin–Madison
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Variable analysis
independent variables
- Rat age
- Rat treatment
- Percentage of each MHC isoform per lane
- Protein concentration of each TA muscle was optimized so that 0.4 μg of protein was added to each gel channel in duplicate
- SDS-PAGE was performed on each concentrate with a 0.75 mm thick 6% acrylamide/30% glycerol separating gel (18 × 16 cm) and a 4% acrylamide/30% glycerol stacking gel
- A silver staining kit (SilverQuest Silver Staining Kit, Life Technologies, New York, USA) was used to stain the gel for visualization of protein bands
- An investigator masked to rat age and treatment determined the optical density of each band using computer-assisted image analysis and densiometry (UN-Scan-IT gel Version 6.1, Silk Scientific, Inc, Utah, USA)
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