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Yu-li Wang's Laboratory: Lab Protocols

Preparation of Gizzard Myosin

Day 1

Materials (all solutions at 4oC)

1. 50 mM KCl, 25 mM MgCl2, 2 mM EGTA, 3% (v/v) Triton-X 100, 0.2 mM DTT, 10 mM Tris-HCl, pH 7.5. Need 9 ml for each gram of tissue.

2. 100 mM KCl, 2 mM EGTA, 0.2 mM DTT, 10 mM Tris-HCl, pH 7.5. Need 9 ml for each gram of tissue.

3. Extraction buffer: 5 mM ATP, 4 mM EDTA, 2 mM EGTA, 0.5 mM DTT, 40 mM imidazole, pH 6.8. Need 1.5 ml for each gram of tissue.

4. PBS, solution A, 2 liters (for thawing gizzards).

5. Meat grinder, prechilled in a cold room and rinsed with 20 mM EDTA, pH 7.0 immediately before use.

6. Scalpels.

7. Turkey gizzards, fresh or frozen at -80oC. It is most convenient and efficient to handle ~200g from 4-5 gizzards.

8. GSA bottles, a lot of. Chill down on ice.

9. 1 M MgCl2, 100 ml.

10. 100 mM ATP, pH 7.0, 10 ml.

11. Polytron, chill down the large generator.

12. Glass wool and funnel.

13. High salt (e.g. 3 M KCl) for soaking electrode.>

14. Large ultracentrafuge rotor and tubes (bottles), e.g. Type 35 or 50.2Ti or 60T.

Procedure

1. Wash/rinse turkey gizzards in cold PBS. Trim gizzards with scalpels to remove fat and connective tissue and cut gizzards into small pieces. To use frozen gizzards, thaw in a 4 liter beaker containing cold PBS at room temperature, until the gizzards become pliable (about 1-1.5 h).

2. Pass dissected gizzards twice through a meat grinder, using first the coarse then the fine mesh. Weigh the tissue.

3. Mix tissue with 3 volumes of buffer 1 in a 2 liter beaker. Homogenize with a Polytron 4 x 15 sec. It gets very foamy but do not worry.

4. Centrifuge in a GSA rotor for 5 min at 4,000 rpm, 4oC.

5. Resuspend pellets in 3 volumes of buffer 1. Homogenize with a Polytron 1 x 20 sec.

6. Repeat steps 4 and 5, then centrifuge as in step 4.

7. Resuspend pellets in 3 volumes of buffer 2. Homogenize with the Polytron 1 x 20 sec.

8. Centrifuge in a GSA rotor for 10 min at 9,000 rpm, 4oC.

9. Perform steps 7 and 8 two more times.

10. Resuspend pellets in 1.5 volumes of extraction buffer. Blend with the Polytron 2 x 20 sec, then adjust the pH to 6.9. The solution is quite viscous and needs to be stirred with a glass rod. Soak the electrode in high salt after this step.

11. Stir by hand continuously on ice for 15 min.

12. Centrifuge in a GSA rotor at 12,000 rpm, 4oC for 20 min.

13. Filter the supernatant through glass wool placed in a funnel. The filtrate may appear slightly turbid. Adjust the pH to 7.6 with 0.1 N KOH and measure the volume.

14. Add 1 M MgCl2 dropwise while stirring the supernatant to obtain a final concentration of 150 mM MgCl2 (0.177 volume of step 13). A peristaltic pump may be used. Then add 100 mM neutralized ATP to a final concentration of 2.5 mM (0.03 volume of step 13). The pH should be monitored and maintained at 7.6 with 0.1 N KOH immediately upon the addition of MgCl2. Failure to do this may result in a reduced yield. Soak the electrode in high salt after this step.

15. Stir gently on ice for 10 min.

16. Centrifuged in a GSA rotor at 12,000 rpm, 4oC for 10 min.

17. Collect supernatant and centrifuge in a Type 35 rotor at 31,000 rpm, 50.2Ti rotor at 29,000 rpm or 60Ti rotor at 32,000 rpm overnight.

Day 2

Materials

1. 1.5 mM EGTA, 5 mM ATP, 0.5 mM DTT, 10 mM (NaH2PO4+Na2HPO4), pH 7.6. Need ~0.5 ml for each gram of tissue.

2. 0.5 M KCl, 1 mM DTT, 10 mM Tris-HCl, pH 7.5, 1 liter.

3. Cold distilled H2O, 10 liters.

4. 1 M MgCl2, 20 ml (from Day 1).

5. 40 ml Dounce homogenizer.

6. High salt (e.g. 3 M KCl) for soaking electrode.

7. 50.2Ti or 60Ti rotor and tubes.

Procedure (performed at 4oC)

1. Collect supernatant and measure the total volume. Dilute slowly with 10 volumes cold distilled water while stirring.

2. Centrifuge in a GSA rotor at 10,000 rpm for 10 min. Two loadings are required for a 200 g preparation, or set up a second Sorvall centrifuge.

3. Resuspend pellets in ~10 pellet volumes of buffer 1. Since the pellets stick tightly on the bottle, they may need to be gently teased with a glass rod then blown off with the buffer using a large-bore pipette.

4. Homogenize gently with a chilled Dounce homogenizer, using slow passage with the A-pestle.

5. Measure volume and adjust pH to 7.6 with 0.1 N KOH. While stirring and monitoring the pH, add dropwise 1 M MgCl2 to obtain a final concentration of 150 mM MgCl2 (volume x 0.177). Soak electrode in high salt after this step.

6. Centrifuge in a 50.2Ti rotor at 40,000 rpm (or a 60Ti at 45,000 rpm) for 2-3 h.

7. Collect supernatant and measure the volume. Dilute with 10 volumes of cold distilled water while stirring. Stir gently on ice for 10 min.

8. Centrifuge in a GSA rotor at 10,000 rpm for 10 min.

9. Resuspend pellets in ~10 pellet volumes of buffer 2 (see steps 3 and 4). Record the volume used.

10. Add cold distilled water while stirring to obtain a final KCl concentration of 0.1 M. Volume equals the volume of buffer 2 used in step 9 times 3.9. Stir for 15 min on ice.

11. Centrifuge in a GSA rotor at 12,000 rpm for 15 min.

12. Collect supernatant. Excessive pellet reflects a failure to maintain pH during earlier MgCl2 precipitation steps. Measure volume and dilute 1:1 with cold distilled water while stirring.

13. Add 1 M MgCl2 dropwise while stirring to obtain a final concentration of 10 mM (1/100 current volume) and place on ice for 1 hr. Don't panic, precipitation of myosin takes time.

14. Centrifuge in a SS34 rotor at 11,000 rpm for 15 min.

15. Resuspend pellets in buffer 2 (see steps 3 and 4). Use ~1 ml per 10 g of tissue. Dialyze overnight.

Day 3

Materials

1. 20 mM KCl, 2 mM MgCl2, 1 mM DTT, 10 mM HEPES, pH 7.5. 1 liter.

2. 50Ti rotor and tubes.

Procedure (performed at 4oC)

1. Clarify in a 50Ti rotor at 40,000 rpm for 1 h. Measure protein concentration by Lowry assay.

2. Dialyze against buffer 1 overnight. The solution turns cloudy due to myosin self-assembly. Store as aliquots in liquid nitrogen.


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