Cytoskeleton isolation

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Procedure

1. Harvest 2 x 10⁷ cells.


2. Add 1 ml Lysis buffer.


3. Sit on ice for 2 min.


4. Spin in microfuge at 15k x 5 min.


5. Wash pellet with 0.5 ml Lysis buffer and then 0.5 ml Wash buffer.

1. Resuspend pellet in 50 µl Release buffer.


2. Incubate on ice for 5 min.


3. Spin in microfuge 15K x 5 min.


4. The supernatant contains myosin released from cytoskeletons. For Western blot, 1/5 of the supernatant is plenty.

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Materials

• Wash buffer
  • 100 mM Pipes pH 6.8
  • 2.5 mM EGTA
  • 1 mM MgCl₂
  • 10 mM TAME
  • 20 mM Benzamine
  
  
• Lysis buffer
  • Wash buffer + 0.5% Triton
  
  
• Release Buffer
  • Wash buffer + 200 mM KCl + 2 mM ATP

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