| |
A. Day 1 - work at 4°C (in a cold room)
1. Obtain fresh rabbit muscle or fresh chicken muscle and cool it on ice.
2. Grind the muscle twice in a meat grinder using a fine mesh for the second grinding. Weigh out approximately 300 to 400 grams of ground muscle.
3. Add 3 volumes of extraction buffer and stir it constantly for 10 min at 4°C.
4. Centrifuge in a pre-chilled GSA rotor for 15 min at 12,000 rpm (23,000 X g) at 4°C. (See Hint #1)
5. Recover the supernatant and adjust its pH to 6.6 slowly with 0.5 M acetic acid. Do this carefully with adequate stirring to avoid myosin precipitation. (See Hint #2)
6. Measure the volume of the solution and dilute it 10-fold with cold ddH2O. Let the cold ddH2O flow very slowly into the beaker with stirring. Check the pH and readjust to 6.6 if necessary.
7. Let the precipitates settle and siphon off the liquid.
8. Alternatively, pellet the precipitated myosin by centrifugation at 7,000 rpm (8,000 X g) in a GSA or GS3 rotor at 4°C for 5 to 15 min.
9. Resuspend the pellet in Buffer 8 with a stirring rod and rubber scraper. Use a total of 0.25 ml for each gram of ground muscle. Save a small portion of the buffer for rinsing out the centrifugation bottles.
10. Dialyze overnight against Buffer 9. Pour the solution directly through a funnel into dialysis tubing and dialyze against a volume that is at least 24-fold greater than the myosin solution volume.
B. Day 2 - work at 4°C (in a cold room)
1. Measure the volume of the dialyzed myosin solution. Add an equal volume of cold ddH2O very slowly with constant stirring.
2. Allow the solution to stir on ice for 30 min.
3. Centrifuge in a SS34 rotor for 30 min at 4°C at 18,000 rpm (39,000 X g).
4. Measure the volume of the supernatant (the pellet contains actomyosin). Dilute the supernatant carefully with 7 volumes of cold ddH2O.
5. Centrifuge the solution in a GSA rotor for 15 min at 12,000 rpm (23,000 X g) at 4°C and discard the supernatant.
6. Resuspend the pellet in a small amount (10 to 15 ml) of 2 M KCl. Measure the volume added. Use a glass stirring-rod to disperse the pellet.
7. Transfer the slurry into a graduated cylinder and calculate the volume of the pellet.
8. Add more 2 M KCl to bring the KCl concentration to 0.5 M. (See Hint #3)
9. Add a minimal volume of 0.5 M KCl to get the pellets into solution.
10. Slowly add 0.67 volume of saturated Ammonium Sulfate and maintain constant stirring.
11. After 15 min, centrifuge in a SS34 rotor for 10 min at 4°C ay 13,000 rpm (20,000 X g).
12. Collect the supernatant and measure its volume. Add 0.2 volume of saturated Ammonium Sulfate (i.e. to 50% saturation).
13. This myosin solution can be stored in 50% saturated Ammonium Sulfate for several months at 4°C. Expect a yield between 1.0 to 1.5 g of myosin from 300 to 400 g of muscle.
|
| 0.5 M KCl |
 |
| 2 M KCl |
|
| Saturated Ammonium Sulfate |
| Bring up to 500 ml ddH2O
Add EDTA to 10 mM and place at 4°C overnight.
Adjust the pH to 8.2 with Ammonium Hydroxide.
Heat until dissolved.
If there are no solid Ammonium Sulfate crystals in the bottom of the solution, add solid Ammonium Sulfate until this occurs.
Add 390 g ultrapure Ammonium Sulfate
Dilute a small amount 1:10 in water and check the pH. It should be 7.0.
|
|
| 1 M KCL |
| For rinsing the pH meter probe
|
|
| 0.5 M Acetic Acid |
|
| Buffer 9 |
| pH 6.5
1 mM DTT
25 mM KH2PO4
10 mM EDTA
0.6 M KCl
Store at 4°C
|
|
| Buffer 8 |
| pH 6.5
1 M KCl
Store at 4°C
60 mM KH2PO4
25 mM EDTA
|
|
| Extraction Buffer |
| pH 6.5
0.3 M KCl
1 mM ATP
0.15 M KH2PO4
5 mM MgCl2
20 mM EDTA
Store at 4°C
|
|
| 100 mM EDTA, pH 7.0 |
|
| Potassium Phosphate Buffer (1 liter) |
| pH 6.5
272 g Potassium Phosphate Monobasic (KH2PO4)
348 g Potassium Phosphate Dibasic (K2HPO4)
|
|
|
| |
ATP
DTT
Potassium Chloride
Magnesium Chloride
EDTA
Ammonium Sulfate
Potassium Phosphate Dibasic
Acetic Acid
Ammonium Hydroxide.
Potassium Phosphate, Monobasic
|
| |
1. The pellet can be used for the production of acetone powder (see protocol).
2. Leave the pH electrode in 1 M KCl for several hr before cleaning up.
3. The actual volume (in ml) should be 0.307 multiplied by the volume of the pellet minus the volume of added KCl.
|
| |
1. Pollard TD. Myosin purification and characterization. Methods Cell Biol 1982; 24:333-71
2. Margossian SS, Lowey S. Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol 1982; 85 Pt B:55-71
|
