is taken from the method of Schafer et
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).
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
and mix by homogenisation.
Stir on ice for 30 minutes.
Centrifuge at 25,400g (15krpm, JA20) keep supernatant.
Centrifuge at 100,000g (35Krpm, Ti45) for 1 hour.
Dialyse against 1 litre of buffer D overnight:-
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.
Spin the dialysate at 25,400g (15krpm, JA20) for 30 minutes to
material after the dialysis, and apply to the DE53 column.
Elute off proteins with a 100-500mM KCl gradient.
Identify actin by 10% SDS-PAGE.
Apply the pooled fractions from the DE53 column onto the DNAse1
column in buffer G (see below)
Wash the column in buffer G, then apply buffer G with 10%
Apply Buffer G with 10% formamide and 0.2M NH4Cl
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.
Gel filter the actin on S-300 or S-200 in buffer G.
to Maciver Lab Protocols
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.
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;
D.J., Eisenberg, E., Korn, E.D. (1976). Characterization of cytoplasmic
actin isolated from Acanthamoeba
castellanii by a new method. J.Biol.Chem.
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