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Protein purification: tropomyosin

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822

 

   
 
  Overview
  TROPOMYOSIN

An ubiquitous actin binding protein arranged in alpha-helical coiled-coil of 410 Angstrom length.

 
 
  Material
  Source: rabbit skeletal muscle

Equipment:
• hydroxylapatite column (2.5 x 25 cm)
• electrophoretic gels
• centrifuge

Chemicals: KCl, Trizma Base, KH2PO4, DL-DTT, 2-mercaptoethanol, imidazole, (NH4)2SO4

column materials: hydroxylapatite.

Have ready:
extraction buffer: 10 mM Tris-HCl, 0.5 mM 2-mercaptoethanol, pH 8.0 (0.2 liter).

imidazole buffer: 2 mM imidazole, 5 mM 2-mercaptoethanol, pH as made (2 liter).

phosphate buffer: 1 M KCl, 1 mM KH2PO4, 2 mM DTT, pH 7.0 (5 liter).

buffer G: 2 mM Tris-HCl, 0.2 mM CaCl2, 0.2 mM ATP, 0.2 mM DTT, 0.05% NaN3, pH 8.0 (15 liter)

 
 
  Procedure
  FLOW CHART

1. obtain acetone powder from rabbit skeletal muscle (10 gram)
[refer to Protein purification; actin (http://iprotocol.mit.edu/protocol/299.htm )]

2. at 0oC, extract in buffer G (2X); 20 ml/gram acetone powder; filter (2X)

3. (take residue) at 25oC, resuspend in 170 ml extraction buffer for 3 h; centrifuge at 16,000 x g for 1 h

4. (take supernatant) cool to +4oC; add 20 mg (NH4)2SO4 per 100 ml; stir for 20 min, centrifuge at 16,000 x g for 30 min

5. (take supernatant) add 30 gram (NH4)2SO4 per 100 ml; stir for 20 min; centrifuge at 16,000 x g for 30 min

6. (take pellet) resuspend in 20 ml imidazole buffer; dialyze against imidazole buffer, for 12 h; centrifuge at 80,000 x g for 2 h.

7. (take supernatant) add KCl to a final concentration of 1M KCl

8. chromatograph on hydroxylapatite column

9. elute with 1 liter linear gradient of 1-200 mM KH2PO4 -->Tropomyosin

 
 
  Troubleshooting
   
 
  Reference
  Smillie, L.B. (1982) Methods Enzymol. 85, 234-241.

Goldmann, W.H. (2000) Biochem. Biophys. Res. Comm. 276, 1225-1228.  

 

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