Isolation of Actin and Myosin Filaments

Isolation of Actin and Myosin Filaments

LEVEL III

Materials

 

• Relaxing Solution
  
  • 0.1 M KCl
    
  • 0.001 M MgCl
    
  • 5 mM ATP
    
  • 0.016 M NaH PO
    
  • Na HPO
    
  • Adjust pH to 7.3
    
• 0.05 M Sodium phosphate buffer, pH 7.0
  
• 0.001 M EDTA (Ethylene diamine tetra acetic acid)
  
• Blender
  
• Preparative centrifuge
  
• Materials for TEM fixation, embedding and observation

Procedure 7

 

1. Obtain fresh chicken gizzards 8 and dissect the lateral muscles free from all attachments. Place the muscles on crushed ice and then grind them in a standard worm- drive meat grinder. Small samples can be pushed through a hand press, if desired.

    

2. Weigh the tissue and add an equal volume of cold 0.05 M Sodium phosphate buffer, pH 7.0 with 0.001 M EDTA. Blend this mix in a standard blender at low speed and pour the slurry into a large beaker.

    

3. Upon settling, the muscle fragments will settle on top of the underlying connective tissue. Separate the two by decanting, and concentrate by low speed centrifugation.

    

4. Suspend aliquots of blended muscle in two volumes of relaxing solution and homogenize in a blender at high speed for 30 seconds.

    

5. Centrifuge the homogenate at 500 xg to remove membraneous organelles and whole cells. The myofilaments are preferentially localized in the middle, clear solution of the centrifuge tube.

    

6. Collect the middle layer and recentrifuge at 40,000 xg to pellet the myofilaments. Wash and gently resuspend. This will rid the preparation of soluble proteins.

    

7. Prepare a small sample of the middle layer from Step 5 for EM observation, following the procedure outlined above for tubulin ( Exercise 9.7 ).