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Please direct all comments, questions, and suggestions to biobzc@hofstra.edu
PREPARATION OF MUSCLE PROTEIN FOR POLYACRYLAMIDE GEL ANALYSIS
Since we know that the common mutant is dominant flightless and has a P-element insertion mutation in the myosin heavy chain gene, it is reasonable to hypothesize that the flight muscle of the mutant flies have less myosin than wild-type flies. To test this hypothesis, you can isolate the flight muscle from mutant and wild type flies and check the amount of myosin protein obtained on an polyacrylamide gel.
• Dissect the muscle from the thorax of 5 wild-type adult flies and 5 adult common mutants. Weigh the tissue and adjust samples so they are equal in weight. Homogenize lightly in a 1.5 ml microcentrifuge tube using a small disposable tissue grinder. Place the homogenate in York Modified Glycerol Solution at -20oC for at least two days.
• Pellet the tissue by centrifugation and remove the supernatant. Re-suspend the tissue in Rigor Buffer with Triton X-100, homogenize lightly. Do this three times.
• Rinse 2 X in Rigor Buffer without Triton X-100.
• Remove buffer and re-suspend the tissue in 20 µl Sample Buffer + BME (beta-mercaptoethanol)
• Heat at 95oC for 5 minutes, load entire sample into one well of a polyacrylamide gel.
York Modified Glycerol
20 mM Na-phosphate buffer, pH 7.0
50% glycerol
0.5% Triton X-100
2 mM MgCl2
1 mM NaN3
1 mM DTT
Rigor Buffer
10 mM Na-phosphate buffer, pH 7.0
100 mM NaCl
2 mM MgCl2
2 mM EGTA
0.1 mg/ml soybean trypsin inhibitor
1 mM DTT
with and without 0.5% Triton X-100