Protein purification; actin

Protein purification; actin      

Overview   ACTIN
The most abundant muscle and non-muscle cytoskeletal protein. MW 42 kDa, 374/375 amino acids; various isoforms; structure resolved at 2.8 Angstrom. [see also protocol ID 207,208,209]     

Material  

1. Source:
rabbit skeletal muscle

2. Equipment:
• mincer
• glass-Teflon-homogenizer with pestle
• sephacryl S-300 column (5 x 100 cm)
• electrophoretic gels
• centrifuge
• sterile cheesecloth


3.Chemicals:
KCl, EDTA, acetone, Trisma Base, CaCl2, MgCl2, ATP, DTT, NaN3; column material: sephacryl S-300

4. Have ready:
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. take ~350 gram rabbit skeletal muscle

2. extract at +4 oC with 0.1 M KCl and filter

3. (take residue) extract at +4 oC with 50 mM NaHCO3 and filter

4. (take residue) extract at +4 oC with 1 mM EDTA, pH 7.0 and filter

5. (take residue) extract at +4 oC with H2O (2x) and filter

6. (take residue) extract at +20-25 oC with acetone (5x) and filter

7. (use muscle acetone powder) extract with buffer G at 0 oC; 20 ml/7 gram acetone powder; centrifuge at 35,000 x g for 30 min.

8. (use combined filtrate) filter through sterile cheesecloth at +4 oC

9. (use supernatant) add 50 mM KCl, 2 mM MgCl2, 0.5 mM ATP for polymerization; incubate for 30 min; stir for 30 min; adjust to 0.8 M KCl; centrifuge at 100,000 x g for 2 h

10. (take muscle F-actin; not supernatant)
homogenize in 300 ml buffer G at +4 oC; dialyze suspension against buffer G for 48 h; centrifuge at 100,000 x g for 2 h

11. (take muscle G-actin; not pellet) chromatograph on sephacryl S-300 column at +4 oC

12. pool G-actin fractions at +4 oC (~10 mg G-actin/gram acetone powder)

 

Reference   ?Sheterline, P. Actin Profile Vol. 1, 1 (1994)Academic Press, London, UK.
?Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F. and Holmes, K.C. Nature (1990) 347:37-44.
?Goldmann, W.H. Biochem. Biophys. Res. Commun. (2000) 271:553-557.