An optimized procedure for isolation of rodent and human skeletal muscle sarcoplasmic and myofibrillar proteins
2Department of Cell Biology and Physiology, Edward Via College of Osteopathic Medicine Auburn Campus, Auburn, AL 36849, USA
3LaGrange College, LaGrange, GA 30240, USA
4School of the Environment, Florida A&M University, Tallahassee, FL 32307, USA
INTRODUCTION
MATERIALS AND METHODS
Ethical approval for rodent study and gastrocnemius dissections
Ethical approval for obtaining human muscle
Experiment 1 procedures
Protein concentration determination
SDS-PAGE and Coomassie staining for Experiment 1
Western blotting
Experiment 2: development of an optimized procedure from Experiment 1 results
Statistics
RESULTS
Protein yields in Experiment 1
Actin and myosin determination by Coomassie staining in Experiment 1
Western blotting results for Experiment 1
Experiment 2: an optimized approach for isolating myofibrillar and sarcoplasmic protein fractions from human muscle samples
DISCUSSION
Method | Pros | Cons |
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GCL |
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Trizol |
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MF method 1 |
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MF method 2 (addition of spermidine to buffer 3 in MF method 1) |
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MF method 3 |
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Optimized method (Experiment 2); recommended |
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Step | General notes | Buffer recipe |
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1 | Obtain 10–50 mg of powdered muscle on a LN2-cooled stage, and place in a 1.7 ml microtube (Tube 1) on a pre-tared analytical scale; weigh tissue for standardizing protein results | N/Aa |
2 | Place Tube 1 with muscle on ice and add 10 volumes (100–500 µl) of ice cold buffer 1 | Buffer 1: 25 mM Tris, pH 7.2, 0.5% Triton X-100 |
3 | Use a tight-fitting microtube pestle to homogenize sample into a slurry, and place Tube 1 back on ice until all samples are homogenized | N/Aa |
4 | Centrifuge Tube 1 at 1500 g for 10 min at 4°C | N/Aa |
5 | Pipet off supernatant (sarcoplasmic fraction) and place into a new 1.7 ml microtube (Tube 2), but leave ~10 µl of supernatant on top of protein pellet in order to reduce sarcoplasmic contamination with MF protein or loss of MF protein; store Tube 2 at −80°C for downstream assays | N/Aa |
6 | Resuspend protein pellet in Tube 1 with 10 volumes (100–500 µl) of ice cold Buffer 1 as a wash step | N/Aa |
7 | Centrifuge tubes at 1500 g for 10 min at 4°C | N/Aa |
8 | Pipet off supernatant, but leave ~10 µl on top of protein pellet in order to reduce loss of MF protein; allow pellets to dry on ice, or use a fine-tip pipettor (e.g., Western blotting loading tip) to remove the remainder of the wash buffer in order to prevent disrupting the MF pellet | N/Aa |
9 | Add 15 volumes of ice cold buffer 2 to pellet in Tube 1, and resuspend using a tight-fitting pestle; remaining/un-suspended protein is putatively collagen | Buffer 2: 20 mM Tris-HCl, pH 7.2, 100 mM KCl, 20% glycerol, 1 mM DTT, 50 mM spermidine |
10 | Perform a quick-spin on a desktop centrifuge for 1 min, and transfer supernatant (MF fraction) to a new tube (Tube 3) | N/Aa |
Notes: |
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Acknowledgments
References
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