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| Author Information | |
| Primary Author | |
| Wolfgang H. Goldmann ( goldman@helix.mgh.harvard.edu ) | |
| Affiliation | |
| Mass. General Hospital , United States | |
| Co-Author(s) | |
| Protocol Information | |
| Protocol ID / PID | |
| PUB299 | |
| Category | |
| Biochemistry | |
| Title | |
| 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) | |
| Troubleshooting | |
| 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. | |
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