Actin Purification Using DNase1 Column Chromatography

This is taken from the method of Schafer et al, 1998. This is a combination of other methods such as the dissociation of actin complexes with high Tris concentrations (Pinder et al, 1995) anion-exchange chromatography on ATP saturated DEAE (Gordon et al, 1976) and DNase1-agarose affinity chromatography (Kron et al, 1992).

1. Take 10g Cells and homogenise in 100mls of extract buffer :-

Extraction Buffer.

Working Concentration	Stock	/100mls
1M Tris-HCl pH 7.0	2M	50mls
0.6M KCl	dry	4.47g
0.5mM ATP	100mM	0.5mls
1mM DTT	100mM	1ml
0.5mM MgCl₂	1M	0.5mls
0.1mM PMSF	100mM	100µls

2. As soon as the cell extract is thawed and mixed by homogenisation add Triton X-100 to 4% (4mls/100mls) and Tween-20 to 1mg/ml (0.1mls/100ml) and mix by homogenisation.

3. Stir on ice for 30 minutes.

4. Centrifuge at 25,400g (15krpm, JA20) keep supernatant.

5. Centrifuge at 100,000g (35Krpm, Ti45) for 1 hour.

6. Dialyse against 1 litre of buffer D overnight:-

Buffer D.

Working Concentration	Stock	/litre
10mM Tris-HCl pH8.0	1M	10ml
0.2mM CaCl₂	100mM	2mls
0.5mM ATP	100mM	5mls
0.1mM DTT	100mM	1ml
100mM KCl	dry	7.45g
0.1mM PMSF	100mM	1ml

7. Equilibrate a DE53 column in buffer D overnight. Add 1.5g of solid ATP per 300mls wet weight of settled DE53, pH immediatley to 8.0. incubate 15 minutes with occasional mixing. Wash with buffer D.

8. Spin the dialysate at 25,400g (15krpm, JA20) for 30 minutes to remove precipitated

material after the dialysis, and apply to the DE53 column.

9. Elute off proteins with a 100-500mM KCl gradient. Identify actin by 10% SDS-PAGE.

10. Apply the pooled fractions from the DE53 column onto the DNAse1 column in buffer G (see below)

Buffer G.

Working Concentration	Stock	100x Stock solution, 100mls
2mM Tris, pH8.0	1M	20mls
0.2mM ATP	0.1M	20mls
0.5mM DTT	dry	0.77g
0.2mM CaCl₂	0.1M	*
0.02% NaN₃	dry	2g
* Do not add CaCl₂ to the 100x buffer as it will precipitate, add when 1x (2mls/litre)

11. Wash the column in buffer G, then apply buffer G with 10% formamide.

12. Apply Buffer G with 10% formamide and 0.2M NH₄Cl pH8.0.

13. The actin should now be eluted off the column but it is vital that the formamide be removed as soon as possible. Collect the actin in tubes that contain the same amount of volume of buffer G as the fraction volume so that the formamide in diluted rapidly to 25%. As soon as the actin is off (monitor by OD₂₉₀), concentrate and wash the actin by using Centricon filter units and buffer G.

14. Gel filter the actin on S-300 or S-200 in buffer G.

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References:-

Schafer, D.A., Jennings, P.B. and Cooper, J.A. (1998). Rapid and efficient purification of actin from non-muscle sources. Cell Mot.Cytoskel. 39; 166-171.

Pinder, J.C., Sleep, J.A., Bennett, P.M. and Gratzer, W.B. (1995). Concentrated Tris solutions for the preparation, depolymerization, and assay of actin: Application to erythroid actin. Anal.Biochem. 225; 291-295.

Gordon, D.J., Eisenberg, E., Korn, E.D. (1976). Characterization of cytoplasmic actin isolated from Acanthamoeba castellanii by a new method. J.Biol.Chem. 251; 4778-4786.

Kron, S.J., Drubin, D.G., Botstein, D. and Spudich, J.A. (1992). Yeast actin filaments display ATP-dependent sliding movement over surfaces coated with rabbit muscle myosin. Proc.Nat.Acad.Sci.USA 89; 4466-4470.