ACTIN EXTRACTION FROM ACETONE POWDER (adopted by Louise Wang and Phillip Allen from Pardee and Spudich. 1982. Purification of muscle actin. Methods Cell Biol. 24:271-289)

Preparation: Wash 2 GS-3 and 6 Type 45TI centrifuge tubes; Reserve centrifuges; Collect beakers, cheese cloth, etc. Make 4L G-buffer right before with cold distilled water, pH before adding DTT since DTT effects the accuracy of the electrode and loses its activity over time.

1. Add 250 ml 1x G-buffer (2 mM Tris, 0.2 mM CaCl2, 0.5 mM ATP, 0.2 mM DTT, pH8.0) to 7-10 g acetone powder in 1L beaker. Stir in cold room for 30' with metal mechanical stirrer at dial 2.5.

2. Filter with 4 layers of cheesecloth fixed onto the top of a plastic beaker with elastic band, but do not squeeze. Label the beaker sup1.

3. Repeat step 1 and 2 by adding 150 ml G-buffer. Label the beaker sup2. The buffer volumes vary as long as sup1 and sup2 can each fit into three 45TI tubes in step 7(Important detail!). Be careful to keep supernatant 1 and 2 separate all the time.

4. Centrifuge the supernatant in Sorvall RC-5C with GS-3 rotor for 30' at 9,000rpm (16,000g) @ 4oC. Pour supernatant into graduate cylinder and read volume. To prevent loose pellets from falling into supernatant, place a piece of cheesecloth over graduate cylinder while pouring supernatant.

5. Add KCl to 50mM and MgCl2 to 2mM (per 100ml of supernatant add: 1.63ml of 3M KCl; 0.2ml of 1M MgCl2). Mix well. Allow to polymerize @ room temperature for 2hrs. Stir gently. Viscosity should increase as evidenced by trapping of air bubbles.

6. Cool for 15' in an ice bucket. Add solid KCl to 0.8M (5.6g/100ml). Stir for exactly 1.5 hr @ 4oC. This step dissociates tropomyosin from actin.

7. Spin for 2 hrs at 40,000rpm in Sorvall Ultra80 @ 4oC. (Next, start the procedures for labeling actin).

8. Decant supernatant thoroughly. Rinse each tube with 1-2ml G(A) buffer to remove any contaminating protein.

9. Note color of actin pellet; it should be transparent and gelatinous. Add 1-2ml G(A) buffer to each centrifuge tube to resuspend the actin pellet (using plastic transfer pipettes). Total volume of resuspension should be 6-9 mls for each sup.

10. Dounce gently on ice to break up the chunks of actin gel.

11. Place into thin dialysis tubes and dialyze for 3 days in 4L beakers of G(A) buffer with changes of buffer daily, and ideally 1-2 hrs before taking the tubes out. Add NaN3 (sodium azide to 0.5-2 mM) to prevent bacterial growth.

12. Centrifuge @ 80k rpm in Sorvsall untracentrifuge (>100k x g) for ?1hr. Keep supernatant and measure OD290 (ex coeff = 0.62).