Protein purification: skeletal muscle myosin
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| Overview | |
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SKELETAL MUSCLE MYOSIN A two-headed hexomeric actin-activated ATPase consisting of two heavy chains of ~200 kDa and four light chains of ~20kDa. The tail portion consists of an alpha-helical coiled-coil rod of 150 nm in length. The most important mechano-enzyme for muscle contraction. |
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| Material | |
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Source: rabbit skeletal muscle Equipment: • centrifuge with GSA-rotor • cellulose nitrate tubes • gauze • DEAE-sephadex column (2.5 x 60 cm) Chemicals: KCl, EDTA, MgCl2, K/PO4, ATP, DTT Have ready: solution A: 0.3 M KCl, 0.15 M potassium phosphate, 0.02 M EDTA, 0.005 M MgCl2, 0.001 M ATP, pH 6.5. solution B: 1 M KCl, 0.025 M EDTA, 0.06 M potassium phosphate, pH 6.5. solution C: 0.6 M KCl, 0.025 M potassium phosphate, pH 6.5. solution D: 0.6 M KCl, 0.05 M potassium phosphate, pH 6.5. solution E: 0.15 M potassium phosphate, 0.01 M EDTA, pH 7.5. solution F: 0.04 M KCl, 0.01 M potassium phosphate, 0.001 M DTT, pH 6.5. solution G: 3 M KCl, 0.01 M potassium phosphate, pH 6.5. |
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| Procedure | |
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FLOW CHART (all procedures are carried out at 4oC) 1. take ~1000 gram skeletal muscle 2. grind twice; extract with 2 liter solution A for 15 min while stiring; add 4 liter cold bidest H2O, filter through gauze (keep residue for actin extraction); dilute with cold H2O to ionic strength of 0.04, (about 10-fold); let settle for 3 h; collect precipitate at 7,000 rpm in GSA rotor for 15 min. 3. (take pellet) disperse in 220 ml solution B; dialyze overnight against 6 liter solution C; slowly add ~400 ml equal volume cold dist. H2O; stir for 30 min; centrifuge at 10,000 rpm for 10 min in GSA rotor. 4. (take supernatant ) centrifuge at 19,000 rpm for 1 h. 5. (take supernatant) dilute to ionic strength of 0.04 (~8-fold); let myosin settle overnight; collect about 5-6 liter fluffy myosin precipitate by centrifuging at 10,000 rpm for 10 min in GSA rotor. 6. (take pellet) resuspend in minimal volume of solution G; dialyze overnight against 2 liter solution D; centrifuge at 19,000 rpm for 2 h, in cellulose nitrate tubes; puncture tubes and separate myosin from fat and insoluble pellet. 7. (take supernatant) [may be stored on ice for 3-4 weeks or in 50% glycerol at -20oC for a year] otherwise dilute to 5-10 mg/ml and dialyze against solution E extensively, load onto DEAE-sephadex column 8. preequilibrate with solution E; apply 500-600 gram myosin at 30 ml/h; wash with 350 ml solution E; elute with linear gradient of 0-0.5 M KCl in solution E (2 x 1 liter); collect 10 ml fractions; pool myosin fractions (>0.1 M KCl); concentrate by overnight dialysis against solution F; centrifuge at 25,000 rpm for 30 min; store as above. -> Myosin |
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| Troubleshooting | |
| Reference | |
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• Margossian, S.S. and Lowey, S. (1982) Methods Enzymol. 85, 55-123. • Goldmann, W.H. and Geeves, M.A. (1991) Anal. Biochem. 192, 55-58. |
