Last updated 8/21/91 jac
Modified from Spudich & Watt, 1971, JBC 246:4866.
1. Mix 20 ml buffer G with each gm of muscle acetone powder. Extract with stirring on ice for 30 min. Use a big stir bar or overhead stirrer. Buffer G: 0.2 mM ATP, 0.5 mM DTT, 0.2 mM CaCl2, 2 mM Tris/HCl, pH 8.0 @ 25°.
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|2 mM Tris/HCl, pH 8 @ 25°|| || || |
|0.2 mM ATP|| || || |
|0.5 mM DTT|| || || |
|0.2 mM CaCl2|| || || |
2. Centrifuge at 16,000 rpm for 30 min in J-20, SS-34 or A600 in cold. If doing as per CapZ prep, filter through cheesecloth as for acetone powder.
3. Filter supernatant thru glass wool layer - coarse & rapid.
4. Resuspend pellet in same volume and repeat steps 2 and 3.
5. Combine the supernatants, stir gently, and make them 50 mM in KCl (2.5 ml of 2 M KCl per 100 ml) and 2 mM MgCl2, (0.2 ml of 1 M MgCl2 per 100 ml).
6. Stir at room temp for 15 min then on ice for 15 min, which polymerizes the actin.
7. While stirring, make 0.8 M in KCl (23.4 ml of 4 M KCl per 100 ml), which dissociates tropomyosin. Stir slowly in cold for 15 min.
8. Centrifuge 2 hrs in Ti 42 or 45 rotor at 35,000 rpm. Discard supernatant. Should see clear/translucent gelatinous pellet. Rinse pellet once with buffer G.
9. Carefully lift entire pellet out of tube by sliding spatula underneath. Transfer to Dounce homogenizer. Rinse remainder of pellet out of tube with buffer G.
10. Suspend pellets in total about 3 ml buffer G per gm of acetone powder and homogenize with Dounce. Should be very viscous, even difficult to transfer to dialysis tubing.
11. Dialyse in thinnest dialysis tubing practical for 2 -3 days. Change the buffer G every day or half day.
12. Spin in Ti 42 or 45 for 2 hrs at 35,000 rpm. Carefully remove top 2/3 of supt. This is "conventional actin." Bottom 1/3 of different tubes can be combined and re-spun, but would not try to make G150 actin from this.
13. Measure concentration by A290. Extinction coefficient is 0.63 ml/mg/cm or 26,600 M-1cm-1.
14. For gel filtration on Sephadex G150, dilute to 5 mg/ml with buffer G. Sample size should be <= 5% of column volume. Collect 4 ml fractions. Column profile should be small peak at void volume, large peak in the back. The large peak should be skewed toward the front. The front of this peak is contaminated with CapZ, so avoid it.
15. Pool fractions and measure concentration. Store by adding 2 mg sucrose per mg actin. Quick-freeze and lyophilize.
16. To use, dissolve and dialyze overnight vs buffer G. In a hurry, you can use the dissolved material without dialysis, but remember that it now has extra ATP from the lyophilized buffer G, so the absorbance is too high.