Dictyostelium myosin isolation (one-day protocol)
Published in Uyeda and Spudich 1993,
Ruppel et al. 1994.
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[MATERIALS] - [INDEX]
Procedure
Note: This protocol involves rounds of myosin assembly and disassembly. Myosins capable of
forming
filaments, therefore, are crucial for a high yield. Cells growing at log-phase are essential for
obtaining active myosin. All procedures after step 1 are performed at 0-5°C.
- Harvest cells.
- Wash in 10 mM Tris-HCl, 1 mM EDTA. Weigh cell pellet.
- Resuspend the cell pellet in 4 vol/g cells of Lysis buffer.
- Add 4 vol/g cells of lysis buffer containing 1% Triton X-100 and a Cocktail of proteinase
inhibitors while gentle swirling. Keep on ice for 5 min. Check the lysis under scope.
- Spin 20k x 30 min in SS34 rotor, discard the supernatant by hand pipetting.
- Resuspend the pellet in 1 vol/g cells of Wash buffer. Dilute 3 fold with a buffer containing
10 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM DTT.
- Spin 20k x 30 min in SS34, discard the supernatant. Be sure to remove any lipids floating in the
supe.
- Resuspend the pellet in 4 vol/g cells of Extraction buffer.
- Spin 52k x 30 min in Ti70 rotor. Remove lipids (if present) in the upper portion of the supe.
- Dilute the supernatant 4-fold with precipitation buffer containing 20 µg/ml RNAase A
(boiled before use), and incubate 20 min on ice.
- Spin 42k x 60 min in Ti45 rotor.
- Homogenize the pellet in 0.3-0.5 vol/g cells of Extraction buffer.
- Spin 40k x 8 min in Ti70.1 rotor.
- Dilute the supernatant 4-fold with precipitation buffer. Incubate on ice for 20 min.
- Spin 52k x 30 min in Ti70.
- Dissolve the pellet in 10-20 µl/g cells of Myosin solubilizing buffer. Clarify by spinning
at 15k x 5 min in table top centrifuge if necessary.
*To achieve improved yield, steps 14 and 15 can be substituted with the following:
- Dialyze the step 13 supernatant overnight against Dialysis buffer. White precipitate forms.
- Spin 20k x 30 min in SS34.
Phosporylation of myosin by MLCK
Phosphorylation by the Dictyostelium MLCK is sometimes necessary to obtain active myosin
purified by this protocol.
- Add 9 vol Kinase buffer and appropriate amount of recombinant MLCK. Incubate at room temperature for 20 min.
- Add 10 mM MgCl2 and incubate for 20 min on ice. Spin 75k x 15 min in TLA100.3 rotor.
- Dissolve the pellet in myosin storage buffer.
* To get better recovery, the starting myosin concentration should be above 2 mg/ml.
[TOP] [INDEX]
Materials
- Lysis buffer
- 25 mM HEPES pH 7.4
- 55 mM NaCl
- 2 mM EDTA
- 1 mM DTT
Wash buffer
- 10 mM HEPES pH 7.4
- 150 mM NaCl
- 1 mM EDTA
- 1 mM DTT
Extraction buffer
- 10 mM HEPES pH 7.4
- 200 mM NaCl
- 3 mM MgCl2
- 2 mM ATP
- 1 mM DTT
Precipitation buffer
- 10 mM HEPES pH 7.4
- 10 mM MgCl2
- 1 mM EDTA
- 1 mM DTT
Protease inhibitor cocktail
- 1 mM PMSF (from 1 M stock; add 100 µl/100ml)
- 50 µg/ml TPCK (from 50 mg/ml stock; add 100 µl /100ml)
- 15 µg/ml pepstatin (from 10 mg/ml stock; add 150 µl/100ml)
- 10 µg/ml leupeptin (from 10 mg/mlstock; add 100 µl/100ml)
Myosin storage buffer
- 10 mM HEPES pH 7.4
- 200 mM NaCl
- 1 mM MgCl2
- 0.5 mM ATP
- 1 mM DTT
Kinase buffer
- 10 mM HEPES pH 7.4
- 3 mM MgCl2
- 2 mM ATP
- 1 mM DTT
Dialysis buffer
- 10 mM Pipes pH 6.5
- 50 mM NaCl
- 10 mM MgCl2
- 1 mM DTT
[TOP] [INDEX]
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