Purification of muscle actin

Published in Pardee and Spudich 1982.
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[MATERIALS] - [INDEX]

Procedure

Preparation of muscle acetone powder

1. For rabbit, dissect out the back and hind leg muscle, place on ice. Alternatively, get a chicken from poultry store and have them kill it and cut the breast and leg muscle for you.
2. 
   
3. Grind the chilled muscle twice and weigh. This is usually 300 g.
4. 
   
5. Extract for exactly 10 min using 3 volumes of extracting solution with stirring in the cold room. (A electrical stirrer will be best, since the magnetic stir bars are generally not powerful enough.)
6. 
   
7. Spin down the pellet and reextract with the same volume of extrating buffer for 10 min.
8. 
   
9. Spin down the pellet and add 1L of cold dH2O to the pellet, stir and adjust the pH to 8.2-8.5 with sodium carbonate (monitor with pH paper).
10. 
    
11. Spin down the pellet.
12. 
    
13. Repeat step 5 and 6 until the pellet begins to swell (This takes about 3-4 repeats).
14. 
    
15. Add the final pellet to cold acetone and stir vigorously for about 20 min. Spin down the pellet in polypropylene centrifuge tube.
16. 
    
17. Repeat step 7.
18. 
    
19. Spread the final pellet on filter paper and allow it to dry in chemical hood.
20. 
    
21. For rabbit acetone powder, wash with chloroform twice and dry again. (No need for chicken acetone powder since it has less fat.)
22. 
    
23. Store desiccated in the freezer.
24. 
    

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Preparation of actin from acetone powder

Note: Since G-actin is sensitive to proteolytic activity during extraction procedure mostly due to bacterial contamination, it is best to make Buffer A fresh from powder or make a concentrated stock solution, stored frozen and dilute to working concentration before use.

1. Prechill buffer to 0°C on ice water.
2. 
   
3. Mix 10 g muscle acetone powder with 200 ml Buffer A and extract with stirring at 0°C for 30 min.
4. 
   
5. Spin down the pellet and filter supernatant through glass wool into graduated cylinder and measure volume.
6. 
   
7. While stirring in beaker, make 50 mM in KCl, 2 mM in MgCl₂ and 1 mM in ATP.
8. 
   
9. Let stand at room temperature for 30 min, then in cold room for 90 min.
10. 
    
11. While stirring, make 0.6 M in KCl and stir slowly in cold room for 90 min.
12. 
    
13. Centrifuge 3 hours in Beckmann 70 Ti rotor at 35 krpm.
14. 
    
15. Discard supernatant, wash pellet with buffer A once and soften the pellet by soaking in 5-10 ml buffer A for 1 hour.
16. 
    
17. Resuspend pellets in buffer A using a Dounce homogenizer (use Teflon coated rod and transfer the solution with plastic pippet to minimize loss).
18. 
    
19. Dialyze for 3 days with 3 changes of Buffer A.
20. 
    
21. Clarify the G-actin solution by centrifugation in 70.1 Ti rotor at 35k rpm for 3 hours.
22. 
    
23. Measure the actin concentration by reading O.D. 290 (1 mg/ml pure actin: 0.65 O.D. 290).
24. 
    
25. Add sucrose (use best grade) to 2 mg/mg actin and lyophilize the G-actin in aliquots. Lyophilized actin can be stored at -70°C for future use.
26. 
    

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Materials

• Myosin extracting solution (cool to 4°C)
  • 0.5 M KCl
  • 0.1M K₂HPO₄
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• Distilled water (cool to 4°C)
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• 1M sodium carbonate
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• Acetone, reagent grade (cool to 4°C)
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• Chloroform
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• Buffer A
  • 2 mM Tris-Cl pH 8.0
  • 0.2 mM ATP
  • 0.5 mM DTT
  • 0.2 mM CaCl₄
  • 0.01% NaN₃

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