Dictyostelium myosin isolation (three day protocol)

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[MATERIALS] - [INDEX]

Procedure

Day -3

1. Seed 3 L HL5 in 6L flask with cells to minimum density 5 x 10⁴ cells/ml.

Day -1

1. Put 70.1 Ti rotor in cold room.


2. Make:
   • 3 L DB
   • 4 L Assembly buffer
   Store in the cold room.
   

Day 1

1. Count cells. Use at least 3 x 10⁹ cells for prep.


2. Precool J6, Sorvall RC, and Beckman centrifuges and SS34 and 70.1 Ti rotors.


3. Pour cells in 1 L bottles, balance.


4. Spin down cells at 3k rpm in J6 at 4°C for 7 min, brake 8.


5. Dump media, resuspend cells in 500 ml cold DB per liter cells, pool in 2 bottles, balance, spin as before.


6. Repeat DB wash again.


7. Resuspend cells in about 100 ml DB, transfer to cold 50 ml conical tubes, balance, spin as before in J6.


8. Measure cell pellet volume, resuspend in two times cell volume of Sonication buffer.


9. Dilute 10 µl cells in 90 µl DB and examine under hemacytometer.


10. Transfer cells to cold plastic beaker on ice water.


11. Using medium tip set at 30% output, sonicate at 0°C with constant stirring 2x 20 sec allowing 20 seconds rest in between bursts.


12. Check cells for 95% lysis. If not, resonicate.


13. When lysed, transfer to cold oak ridge tubes, balance, and spin at 18k rpm, 30 min in cold SS34 rotor in cold Sorvall.


14. Using Pasteur pipet, pipet low speed supernatant1 into about 4 cold polycarbonate tubes, balance, cap, and spin in 70.1 Ti at 41k rpm (150,000g), 4°C, 2 hr.


15. Using Pasteur pipet, pipet high speed Supernatant 2 to long, wide dialysis tubing allowing volume for 4x swelling. Avoid taking soft pellet 2. Use 1 dialysis tube per 1 polycarbonate tube.


16. Dialyse overnight versus 4 L cold Assembly buffer.


17. Put 70.1 Ti rotor back in cold room overnight.

Day 2

1. Precool Sorvall RC and Beckman centrifuges, SS34 and 70.1Ti rotors, and large Dounce homogeniser.


2. Transfer dialysate to cold oak ridge tubes, balance, and spin 18k rpm, 4°C, 30 min to pellet actomyosin filaments.


3. Prewash superose 6 column with two column volumes (60 ml) Column buffer at less than 0.5 ml/min.


4. Resuspend pellets P3 in about 2 ml total cold TK buffer, pool in Dounce homogeniser on ice, measure volume.

Add equal volume cold 2x Disassembly buffer and homogenise.

5. Transfer homogenate to cold polycarbonate tube, cap, balance, and spin 150,000g (no more than 40k rpm), 4°C, 30 min.


6. While 70.1Ti is spinning preload 4 ml 1x Disassembly buffer on column so that it will be done when spin is completed (about 8 min).


7. Load 2 ml supernatant 4 on column. Postload 1 ml 1x Disassembly buffer. Elute with 30 ml Column buffer.


8. While column is running pour two thick 15 well 10% minigels.


9. Take 10 µl aliquots of every other fraction into labeled tubes containing 10 µl 2x SDS gel sample buffer, mix, boil 5 min, run hot aliquots on gel at 150V.


10. Coomassie stain gels at least 30 min, destain until actin and myosin bands appear. Myosin is in fractions 16-26. Actin appears in fraction 40-50.


11. Pool the myosin fractions without actin (about 8) avoiding the trailing edge.


12. Dialyse myosin pool versus 2 L Dialysis buffer 2.

Day 3

1. Precool Sorvall RC centrifuge, SS34 rotor, rotor adaptor, and 1 ml Dounce homogeniser.


2. Transfer dialysate to cold polycarbonate tube. Measure volume.


3. Add MgCl₂ to 10 mM. Hold at 0°C, 60 min.


4. Prepare standard protein samples for Bradford assay.


5. Balance tube and spin 18k rpm, 4°C, 30 min to pellet myosin filaments.


6. Pour low and high percentage PAGE  minigels.


7. Resuspend Pellet 5 in 1 ml Storage buffer, transfer to homogeniser and homogenise gently. Save supernatant 5!


8. Transfer to cold 1.5 ml eppendorf tube, spin in cold room microfuge at top speed, 15 min.


9. Assay protein standards for standard curve.


10. Transfer clarified Supernatant 6 to fresh cold 1.5 ml tube. This has myosin monomers!


11. Assay aliquots of myosin for protein concentration. If too dilute, redialyse versus 1 L Dialysis buffer 2 and recapture.


12. Run aliquots on PAGE for purity.

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Materials

• Sonication buffer (100 ml)
  
  • 20 ml of 0.2 M tetrasodium Pyrophasphate (Nappi) (40 mM final concentration)
  • 45 g sucrose (45% final concentration)
  • 1 ml of 0.5 M Triethanolamine (Teola) pH 7.5 (5 mM final concentration)
  • 0.1 ml 10% sodium azide (0.01% final concentration)
  
  
  Add fresh:
  
  • 100 µl of 1 M PMSF (1 mM final concentration)
  • 100 µl of 50 mg/ml TPCK (50 µg/ml final concentration)
  • 150 µl of 10 mg/ml pepstatin (15 µg/ml final concentration)
  • 100 µl of 10 mg/ml leupeptin (10 µg/ml final concentration)
  • 500 µl of 1 M DTT (5 mM final concentration)


• Assembly buffer 1 (4 L)
  
  • 12.1 g PIPES, free acid (10 mM final concentration)
  • 7.4 g EDTA (5 mM final concentration)
  • 14.9 g KCl (50 mM final concentration)
  • 0.8g Sodium azide (0.02% final concentration)
  
  
  Adjust pH to 7.0 with ~45 pellet of NaOH, bring to volume, cool down and add fresh:
  
  • 4 ml 1 M DTT (1 mM final concentration)
  • 2 ml 1 M PMSF (0.5 mM final concentration)


• Column buffer (200 ml)
  • 4 ml 0.5 M Teola, pH 7.5 (10 mM final concentration)
  • 0.4 ml 0.5 M EDTA, pH 8.0 (1 mM final concentration)
  • 8.95 g KCl (0.6 M final concentration)
  • 0.2 ml 10% Sodium azide (0.01% final concentration)
  
  
  Bring to volume, cool down, and add:
  
  • 0.4 ml of 0.1 M ATP, pH 7.0 (0.2 mM final concentration)
  • 0.2 ml of 1 M DTT (1 mM final concentration)
  
  Filter through 0.22 µm filter before use


• TK buffer (50 ml)
  • 1ml of 0.5 M Teola pH 7.5 (10 mM final concentration)
  • 0.8 ml of 3 M KCl (50 mM final concentration)


• 2x Disassembly buffer (50 ml)
  • 10g KI (1.2M final concentration)
  • 1 ml of 0.5 M Teola,pH 7.5 (10 mM final concentration)
  • 0.5 ml of 1 M MgCl₂ (10 mM final concentration)
  • 0.1 ml of 0.5 M EDTA, pH 8.0 (1 mM final concentration)
  • 0.1 ml of 1 M CaCl₂ (2 mM final concentration)
  • 0.05 ml of 1 M DTT (1 mM final concentration)
  • 5 ml of 0.1 M ATP (10 mM final concentration)
  
  1x Disassembly buffer can be made by diluting 2x with TK buffer.


• Assembly buffer 2 (1 L)
  • 10 ml of 1 M Tris-Maleate, pH 6.5 (10 mM final concentration)
  • 16.7 ml of 3 M KCl (50 mM final concentration)
  • 1 ml of 1 M DTT (1 mM final concentration)


• Myosin storage buffer (10 ml)
  • 0.2 ml of 0.5 M Teola, pH 7.5 (10 mM final concentration)
  • 0.2 ml of 0.5 M EDTA (10 mM final concentration)
  • 0.8 ml of 3 M KCl (0.25 M final concentration)
  • 10 µl of 1 M DTT (1 mM final concentration)
  • 10 µl of 10% Sodium azide (0.01% final concentration)

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