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Yu-li Wang's Laboratory: Lab Protocols

Extraction of Brain Actin from the Acetone Powder

Day 1

Materials

1. 2 mM Tris-Cl, 0.2 mM ATP, 0.5 mM DTT, 0.02% NaN3, 500 ml pH 7.4 at 4oC and 4-5 liters pH 7.6 at 4oC.

2. 20 mM MES, 50 mM NaCl, 2 mM EGTA, 0.5 mM ATP, 4 M glycerol, pH 6.5 at room temperature, 4 liters.

3. Dounce homogenizer, with "A" and "B" plungers.

4. Sonicator with microtip.

5. Chilled 50.2Ti rotor and tubes. Can be substituted with SS34.

6. Giant and medium dialysis tubing.

7. 4 liter beaker or vessel for large volume dialysis.

Procedure

1. Take 8-10 grams of acetone powder, suspend in 50 volumes of buffer 1, pH 7.4.

2. Homogenize the suspension in a Dounce homogenizer, using "B" plunger.

3. Adjust pH to 7.4 with 0.1 N NaOH.

4. Homogenize with the "A" plunger, adjust pH as in step 3.

5. Sonicate 2 bursts on ice, setting 4 on Branson, 10 sec each. Adjust pH again.

6. Stir for 30 min on ice.

7. Centrifuge at 35,000 rpm for 1 hr, 4oC in 50.2Ti rotor or 18,000 rpm for 1 hr, 4oC in SS34 rotor. Warm up the 50.2Ti rotor after this step.

8. Dialyze the supernatant against 4 liter of buffer 2 at room temperature for 3 hrs. Use giant tubing.

9. Centrifuge in a 50.2Ti rotor at 22,000 rpm for 40 min, room temperature.

10. Soak pellets in 8-10 ml of buffer 1 pH 7.6 at 0oC for 30-60 min. Resuspend pellets and rinse tubes with 2-4 ml buffer. Total volume buffer 2

should be around 12 ml.

11. Dialyze overnight against buffer 2 pH 7.6 at 4oC, change buffer 2 times.

Day 2

Materials

1. Buffer 1, pH 7.6 as for day 1, 2 liters.

2. 2 mM Tris-Cl, 0.5 mM ATP, 0.5 mM DTT, 0.2 mM CaCl2, 0.02% NaN3 titrated to pH 8.0 at 4oC with KOH, 2 liters (buffer A).

3. 3 M KCl.

4. 100 mM MgCl2.

5. Sepharose 6B-Cl column 2.5x90 cm, preequbriated with buffer 1, pH 7.6.

6. 50Ti and 50.2Ti rotors and tubes, chilled.

Procedure

1. Centrifuge 30,000 rpm, 4oC for 30 min in a 50Ti rotor.

2. Load on Sepharose 6B-Cl column, 2.5 cm x 90 cm, elute with buffer 1. Collect 3 ml fractions. Use 0.2 to 0.5 OD scale on the chart recorder.

3. Pool fractions in the 2nd peak, measure volume.

4. Add KCl to 100 mM and MgCl2 to 2 mM, let sit at room temperature for 30 min.

5. Centrifuge at 40,000 rpm for 3 hr in a 50.2Ti rotor, 4oC.

6. Soak pellets in a total of 1 ml buffer A for 1-2 hr, 0oC.

7. Resuspend pellets and dialyze against buffer A for 24 hr at 4oC, change buffer twice.

Day 3

Materials

1. 50Ti rotor and tubes, chilled.

2. Ultrapure sucrose.

Procedure

1. Clarify at 40,000 rpm in a 50Ti rotor, 4oC for 3 hrs.

2. Collect supernatant, measure volume and concentration of actin with Lowry assay.

3. Calculare the total amount of actin, add 2 mg ultrapure sucrose per mg actin and stir gently at 0oC until sucrose dissolves completely.

4. Lyophilize. Store dessicated at -20oC. Expect 5-8 mg from 8 g powder.

Reference

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


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