| Contributor: |
The Laboratory of Timothy Mitchison at Harvard University
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| The key variable in microtubule spindown experiments is ATP. Under high ATP conditions, conventional microtubule associated proteins (MAPs) selectively co-sediment with microtubules. In the absence of ATP (or in presence of AMPPNP, which induces rigor binding of motors to microtubules), both motors and MAPs bind to microtubules and sediment with the microtubules. For experiments on co-sedimentation of an unknown protein with tubulin, it is best to try a variety of conditions. Below is a protocol for tubulin/MAP co-sedimentation under high ATP conditions and MAP/motor/tubulin co-sedimentation. |
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A. MAP Co-Sedimentation
1. Centrifuge the extract at 70,000 rpm in a TLA100.3 rotor (202,500 X g) for 20 min at 4°C. Transfer the supernatant to a tube on ice.
2. Extracts must have a source of GTP (see Hint #1). For Xenopus egg extracts, simply adding an Energy Regeneration Mix allows the extracts to maintain physiological GTP levels. For dilute tissue culture extracts, it is best to supplement the extract with 0.5 mM Mg-GTP. This can be done before or after Step #A1.
3. Add MgATP to 2 mM to the extracts. Add Protease Inhibitor Cocktail to the extract (to a final concentration of 1X).
4. Warm the extracts to room temperature and add Taxol to 5 μM. Mix well.
5. Incubate for 2 to 3 min and then add additional Taxol to give a final concentration of 20 μM Taxol.
6. Incubate for 20 to 30 min at room temperature to 37°C. The physiological temperature for Xenopus extracts is room temperature; for tissue culture cells, Tubulin will polymerize better at higher temperatures. Incubating at 30°C to 33°C is a good compromise between polymerization and proteolysis of Tubulin (see Hint #2).
7. Layer the polymerized mixture onto Sucrose Cushion Solution.
8. Centrifuge at 40,000 rpm in a TLS55 rotor (106,000 X g) for 20 min at 22°C. A fixed angle rotor such as a TLA100.3 or TLA100.4 may also be used (see Hint #3). For larger ultra rotors, increase the speed and/or time to reach an equivalent clearing factor.
9. Save the supernatant for Polyacrylamide Gel and Western Analysis. Aspirate the cushion while washing 2 to 3 times with BRB80 and remove as much of the cushion as possible. The Microtubules form a clear, gelatinous pellet.
10. Boil the pellet in Laemmli Sample Buffer and load on a Polyacrylamide Gel (see Protocol on SDS-PAGE).
B. MAP and Motor Co-Sedimentation with Tubulin (see Hint #4)
1. Centrifuge the extract at 70,000 rpm in a TLA100.3 rotor (202,500 X g) for 20 min at 4°C. Transfer the supernatant to a tube on ice.
2. Extracts must have a source of GTP (see Hint #1). For Xenopus egg extracts, simply adding an Energy Regeneration Mix allows the extracts to maintain physiological GTP levels. For dilute tissue culture extracts, it is best to supplement the extract with 0.5 mM Mg-GTP. This can be done before or after Step #B1.
3. Add Protease Inhibitor Cocktail to the extract (to a final concentration of 1X). Do not add extra ATP.
4. Add 2 to 5 mM MgAMPPNP and/or supplement the extract with 15 Units/ml hexokinase and 20 mM Glucose. It is best to set up tubes for each of three conditions: only AMPPNP, only Glucose-Hexokinase, both AMPPNP and Glucose-Hexokinase present.
5. Warm the extracts to room temperature and add Taxol to 5 μM. Mix well.
6. Incubate for 2 to 3 min and then add additional Taxol to give a final concentration of 20 μM Taxol.
7. Supplement extract with Taxol-stabilized Microtubules to 0.2 to 0.3 mg/ml final concentration (see Section C and Hint #5).
C. Preparation of Taxol Stabilized Microtubules
1. Dilute the Tubulin to 2 mg/ml Tubulin in BRB80 + 1 mM GTP + 1 mM DTT on ice.
2. Incubate 5 min.
3. Warm to 37°C and add 1:100 volume of 0.02 mM Taxol.
4. Incubate 5 min at 37°C.
5. Add 1:100 volume of 0.2 mM Taxol.
6. Incubate 5 min at 37°C.
7. Add 1:100 volume of 2 mM Taxol.
8. Incubate 10 to 15 min at 37°C. These Microtubules can be stored at room temperature for up to 1 week.
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| 5 Units/ml Hexokinase |
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| BRB80 + 1 mM GTP + 1 mM DTT |
| 1 mM DTT
Prepare in BRB80 (1X)
1 mM GTP
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| Laemmli Sample Buffer (2X) |
| 200 mM DTT
20% (v/v) Glycerol
125 mM Tris-Cl, pH 6.8
0.2% (w/v) Bromophenol Blue
4% (w/v) SDS
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| Sucrose Cushion Solution |
| Prepare in BRB80 (1X)
0.5 mM ATP
1X Protease Inhibitor Cocktail
10 μM Taxol
1 M Sucrose
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| 20 mM Glucose |
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| Energy Regenerating Mix (20X) |
| 200 mM Phosphocreatine
Store in 10 and 50 μl aliquots at -80°C
20 mM ATP
20 mM MgCl2
2 mg/ml Creatine Kinase
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| BRB80 (5X) |
| pH to 6.8 with KOH
5 mM MgCl2
400 mM PIPES
Store at 4°C
5 mM EGTA
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| 2 mM Taxol |
| Prepare in anhydrous DMSO
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| 5 mM MgAMPPNP |
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| 0.2 mM Taxol |
| Prepare in anhydrous DMSO
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| 0.02 mM Taxol |
| Prepare in anhydrous DMSO
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| Taxol Stock |
| Prepare in anhydrous DMSO
10 mM Taxol (sold under the tradename "Paclitaxel", Sigma) (CAUTION! see Hint #6)
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| Protease Inhibitor Cocktail (1,000X) |
| 10 mg/ml Pepstatin (CAUTION! see Hint #6)
10 mg/ml Leupeptin (CAUTION! see Hint #6)
Prepare in DMSO (CAUTION! see Hint #6)
10 mg/ml Chymostatin (CAUTION! see Hint #6)
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EGTA
PIPES
DTT
Bromophenol Blue
GTP
SDS
Phosphocreatine
Glucose
Hexokinase
MgATP
Phosphocellulose-Purified Tubulin
Creatine Kinase
Taxol
Sucrose
Glycerol
DMSO
Magnesium Chloride
Leupeptin
Potassium Hydroxide
Chymostatin
Pepstatin
MgAMPPNP
Tris
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1. The contributor of this protocol has used this protocol with extracts prepared with BRB80 and XB (10 mM HEPES, pH 7.7; 100 mM KCl, 50 mM sucrose, and 1 mM Magnesium Chloride - a classically microtubule-unfriendly buffer) and find that it works with both. Try your favorite buffer and BRB80 side-by-side to maximize chances of success. The buffer used in the cushion is not very important.
2. Extracts must be supplemented with a peptide protease inhibitor cocktail just before warming the extract to prevent excessive proteolysis.
3. The contributor suggests centrifuging the microtubules at 100 to 150,000 X g for 20 to 30 min in TLA100.X rotors over a 30 to 40% glycerol or sucrose cushion made up in BRB80.
4. For MAP and motor analysis, the contributor often supplements the extract with exogenous taxol-stabilized microtubules (see Section C) to ensure that binding sites are not limiting, so that the results seen are not due to competition.
5. This concentration is for concentrated Xenopus. Try using 0.1 mg/ml Taxol-stabilized Microtubules for tissue culture cell extracts.
6. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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