Preparation of Brain Acetone Powder

Materials

1. 0.8% NaCl, 0^oC, 1000 ml.

2. 100 mM MES, 1 mM EGTA, 0.5 mM MgCl₂, 0.25 mM GTP, pH 6.5 at 4^oC, 500 ml.

3. Acetone, 12 liters, room temperature.

4. Fine forceps and scissors.

5. Plastic pan, preweighed and chilled.

6. Polytron or Waring blender, prechilled.

7. 50.2 Ti rotor (2 if possible), chilled. Use SS34 if not available.

8. 2-liter side-arm flask.

9. Buchner funnel and Plurostopper.

10. Whatman #1 filter paper to fit the funnel, also large ones for drying the powder.

11. Stirring plates and large bars.

Procedure

1. Obtain 3-4 lamb or cow brains from local slaughter house, chill on ice.

2. Remove blood vessels and meninges while keeping the tissue on ice or in cold room. Collect tissue in a chilled plastic pan.

3. Weigh tissue. Four lamb brains yield ~350 grams.

4. Transfer to a chilled beaker, rinse with ice cold 0.8% NaCl.

5. Homogenize brains in one volume of buffer 2 (45 sec. low speed in a Waring blender, or better use a polytron for 20 sec).

6. Spin homogenate in 50.2 Ti rotor, 35,000 rpm for 1 hr at 4^oC.

7. Resuspend pellets in 10 volumes (based on starting weight) of acetone and stir for 15 sec. Do this in the fume hood.

8. Let solution sit for 15 min at room temperature to allow settlement of powder. Decant acetone as much as possible.

9. Repeat steps 7 & 8 two more times.

10. Filter out acetone under suction.

11. Spread residues on large filter paper. Cover with aluminum foil and allow to dry overnight in the hood. Poke holes on foil to allow evaporation of acetone.

12. Weigh out dried powder. Expect about 40 g from 350 g of brain. Put ~10 g in each zip-lock bag and store dessicated at -70^o.

Reference

M.F.Ruscha and R.H.Himes (1981) A simple procedure for the isolation of brain actin. Prep. Biochem. 11:351-360.