Chromatographic Purification of Actin

(Adapted from Pardee and Spudich. 1982. Purification of muscle actin. Methods Cell Biol. 24:271-289)


DO THE CHROMATOGRAPHY IN 4oC COLD ROOM FOR THE WHOLE TIME. Pack the column (~70-cm length, ~2.7-cm inside diameter, ~300-ml column volumn) with Sephadex G-150 coarse beads. First wash and equilibrate the column with G-actin buffer, then take off the top cover, let the buffer flow till the top surface of the beads gets almost a little dry before loading the sample slowly, drop by drop, by leaning the pipette tip against the wall of the column. When the sample flows off the top of the resin, add some buffer to the top of the beads and then replace the top cover of the column. The tubing from the buffer reservoir should be connected to the top cover.

To find out which fractions have actin: 1) use a UV monitor and a chart recorder; the first peak is usually the major contaminant protein and second peak is where the REAL actin starts to present; 2) do a Bradford assay of every 5th or 10th fraction, plot the protein concentration(mg/ml) vs. fraction number. Run SDS-PAGE analysis of all the fracions within the second peak to check if actin is in them. As a control, pick some fractions outside of the second peak, in the void volumne, to run on a SDS-PAGE.

The flow rate was approximately 44ml/hr. The settings used in our lab were: fraction size 7min, chart speed 0.2mm/min. Pool together the actin-containing fractions. To determine the concentration of actin, measure OD290, the distinction coefficient for G-actin is 0.63. You can further concentrate the actin.

Store the column in 0.05mM Sodium Azide.

Note: if not using a pump, the column should be at least 60cm above the fraction collector to balance the pressure. The buffer reservoir is at 2/3 the height of the column.