Dictyostelium myosin isolation (one-day protocol)

Published in Uyeda and Spudich 1993, Ruppel et al. 1994.

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[MATERIALS] - [INDEX]

Procedure

Note: This protocol involves rounds of myosin assembly and disassembly. Myosins capable of forming filaments, therefore, are crucial for a high yield. Cells growing at log-phase are essential for obtaining active myosin. All procedures after step 1 are performed at 0-5°C.

1. Harvest cells.
2. 
   
3. Wash in 10 mM Tris-HCl, 1 mM EDTA. Weigh cell pellet.
4. 
   
5. Resuspend the cell pellet in 4 vol/g cells of Lysis buffer.
6. 
   
7. Add 4 vol/g cells of lysis buffer containing 1% Triton X-100 and a Cocktail of proteinase inhibitors while gentle swirling. Keep on ice for 5 min. Check the lysis under scope.
8. 
   
9. Spin 20k x 30 min in SS34 rotor, discard the supernatant by hand pipetting.
10. 
    
11. Resuspend the pellet in 1 vol/g cells of Wash buffer. Dilute 3 fold with a buffer containing 10 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM DTT.
12. 
    
13. Spin 20k x 30 min in SS34, discard the supernatant. Be sure to remove any lipids floating in the supe.
14. 
    
15. Resuspend the pellet in 4 vol/g cells of Extraction buffer.
16. 
    
17. Spin 52k x 30 min in Ti70 rotor. Remove lipids (if present) in the upper portion of the supe.
18. 
    
19. Dilute the supernatant 4-fold with precipitation buffer containing 20 µg/ml RNAase A (boiled before use), and incubate 20 min on ice.
20. 
    
21. Spin 42k x 60 min in Ti45 rotor.
22. 
    
23. Homogenize the pellet in 0.3-0.5 vol/g cells of Extraction buffer.
24. 
    
25. Spin 40k x 8 min in Ti70.1 rotor.
26. 
    
27. Dilute the supernatant 4-fold with precipitation buffer. Incubate on ice for 20 min.
28. 
    
29. Spin 52k x 30 min in Ti70.
30. 
    
31. Dissolve the pellet in 10-20 µl/g cells of Myosin solubilizing buffer. Clarify by spinning at 15k x 5 min in table top centrifuge if necessary.
32. 
    

*To achieve improved yield, steps 14 and 15 can be substituted with the following:

• Dialyze the step 13 supernatant overnight against Dialysis buffer. White precipitate forms.
• 
  
• Spin 20k x 30 min in SS34.

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Phosporylation of myosin by MLCK

Phosphorylation by the Dictyostelium MLCK is sometimes necessary to obtain active myosin purified by this protocol.

1. Add 9 vol Kinase buffer and appropriate amount of recombinant MLCK. Incubate at room temperature for 20 min.
2. 
   
3. Add 10 mM MgCl₂ and incubate for 20 min on ice. Spin 75k x 15 min in TLA100.3 rotor.
4. 
   
5. Dissolve the pellet in myosin storage buffer.
6. 
   

* To get better recovery, the starting myosin concentration should be above 2 mg/ml.

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Materials

• Lysis buffer
  • 25 mM HEPES pH 7.4
  • 55 mM NaCl
  • 2 mM EDTA
  • 1 mM DTT

• Wash buffer

• 10 mM HEPES pH 7.4
• 150 mM NaCl
• 1 mM EDTA
• 1 mM DTT

• Extraction buffer

• 10  mM HEPES pH 7.4
• 200 mM NaCl
• 3 mM MgCl₂
• 2 mM ATP
• 1 mM DTT

• Precipitation buffer

• 10 mM HEPES pH 7.4
• 10 mM MgCl₂
• 1 mM EDTA
• 1 mM DTT

• Protease inhibitor cocktail

• 1 mM PMSF (from 1 M stock; add 100 µl/100ml)
• 50 µg/ml TPCK (from 50 mg/ml stock; add 100 µl /100ml)
• 15 µg/ml pepstatin (from 10 mg/ml stock; add 150 µl/100ml)
• 10 µg/ml leupeptin (from 10 mg/mlstock; add 100 µl/100ml)

• Myosin storage buffer

• 10  mM HEPES pH 7.4
• 200 mM NaCl
• 1 mM MgCl₂
• 0.5 mM ATP
• 1 mM DTT

• Kinase buffer

• 10 mM HEPES pH 7.4
• 3 mM MgCl₂
• 2 mM ATP
• 1 mM DTT

• Dialysis buffer

• 10 mM Pipes pH 6.5
• 50 mM NaCl
• 10 mM MgCl₂
• 1 mM DTT

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