Cytoskeleton isolation

Published in Reines and Clarke 1985.

Download word file.

---

[MATERIALS] - [INDEX]

Procedure

1. Harvest 2 x 10⁷ cells.
2. 
   
3. Add 1 ml Lysis buffer.
4. 
   
5. Sit on ice for 2 min.
6. 
   
7. Spin in microfuge at 15k x 5 min.
8. 
   
9. Wash pellet with 0.5 ml Lysis buffer and then 0.5 ml Wash buffer.
10. 
    

The pellet contains actomyosin cytoskeletons. To release myosin from the pellet:

1. Resuspend pellet in 50 µl Release buffer.
2. 
   
3. Incubate on ice for 5 min.
4. 
   
5. Spin in microfuge 15K x 5 min.
6. 
   
7. The supernatant contains myosin released from cytoskeletons. For Western blot, 1/5 of the supernatant is plenty.

[TOP] [INDEX]

---

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

[TOP] [INDEX]

	Return to Home	Supported by NIH (NIGMS and NHGRI)	Send a Message to the dictyBase Curators