Myosin-F-actin binding by pelleting assay

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[MATERIALS] - [INDEX]

Procedure

1. Aliquot 5 µl G-actin or 5 µl of buffer A to individual tubes and polymerize by adding 60 ml of HSB and incubating at 25-37°C for 30-60min.


2. Add 5 µl of 0.2 mM phalloidin.


3. Add 5 µl dextrose.


4. Add 5 µl diluted hexokinase solution to all "ATP minus" samples.


5. Add 5µl of 40 mM ATP to all "ATP plus" samples.


6. Incubate at room temperature for 3-5 min.


7. Add 20 µl myosin and vortex gently.


8. Add 100 µl sucrose cushion (with or without ATP) to TLA100 tube and carefully layer each sample on the cushion.


9. Spin at 75K rpm for 20 min.


10. Remove 125 µl supernatant (include upper portion of the sucrose cushion), TCA precipitate and resuspend in SDS gel sample buffer.


11. Remove rest of the cushion, rinse carefully with dH₂O, resuspend pellet in SDS sample buffer.

[TOP] [INDEX]

Materials

• Buffer A
  • 2 mM Tris-Cl pH 8.0
  • 0.2 mM ATP
  • 0.5 mM DTT
  • 0.2 mM CaCl₄
  • 0.01% NaN₃
  
  
• G-actin
  • 2-4 mg/ml in buffer A
  
  
• Phalloidin
  • 1 mM phalloidin in imidazole-Cl pH 7.0
  • 3 mM NaN₃
  
  Dilute to 0.2 mM before use (1:5).


• 40 mM ATP


• Myosin
  • 1/5-1/10 in molar ratio to actin


• Dextrose
  • 1 M dextrose
  • 3 mM NaN₃


• Hexokinase Sigma H-5875 (4U/ml)
  
  Dilute 20 fold with cold dH₂O before use.


• High salt buffer mix (HSB)
  • 1M KCl
  • 0.17 mM DTT
  • 33.3 mM imidazole-Cl pH 7.0
  • 6.7 mM MgCl₂
  • 3 mM NaN₃


• ATP sucrose cushion (10 ml)
  • 6 ml HSB
  • 3 g sucrose
  • 0.5 ml 40 mM ATP
  
  Bring volume to 10 ml with water.


• Sucrose cushion without ATP
  • same as above, without ATP


• Beckman ultracentrifuge TL-100 with rotor TLA100