Acetone Powder Prep by Schleicher et al. (adapted from thesis of Ludwig Eichinger)

 

 

Prepare one day ahead:

2 L 0.5M KCl; 0.1M K2HPO4 (74.5g KCl, 45.6g K2HPO4.3H2O)

10 L distilled water

1M Na2CO3

5 L cold acetone

meat grinder, reserve large centrifuge

keep everything in cold room

For 300g of muscle:

1. grind two times.

2. extract(dissolve in the solution and stir) for 10 min in 900-ml KCl, K2HPO4

3. spin @ 4000xg for 10min, weigh the pellets.

4. extract pellets again in KCl;K2HPO4

5. Spin @ 4000xg for 10min., weigh the pellets.

6. Add cold water and re-suspend the pellets.

7. Raise pH to 8.2-8.5 with 1M Na2CO3

8. Spin @ 4000xg for 10min.

9. Decant the supernatant and weigh pellet.

10. Re-suspend pellet in water again, check pH to keep it ~ 8.2.

11. Repeat the water extraction until the pellet weight start to increase.

NOTE: The weight of the pellet will go down the first 2 spins or so, and then will begin to rise.

12. After the last water extraction, pool the pellets into one 4L glass beaker and add 4 L cold acetone.

13. Quickly re-suspend and immediately filter through cheesecloth when precipitate starts to form.

14. Repeat once more with acetone.

15. Dry the acetone powder overnight in the hood on filter paper.

To get rid of the lipids in the acetone powder, extract dried powder with chloroform by filtering the chloroform-acetone powder mix through a Büchner funnel sitting over an Erlenmeyer flask. Dry in hood again.