User:Henry Hess/Notebook/Kinesin Kings/2012/06/04
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June 4, 2012: High Density Motility Assay
Name [Stock] Abbreviation BRB80 MgCl2 100mM Mg GTP 25mM GTP DMSO 5% DMSO Rhodamine Tubulin 20µg Taxol 1mM TX Casein 20mg/mL CS D-Glucose 2M DG Glucose Oxidase 2mg/mL GO ATP 100mM ATP Catalase 0.8mg/mL Cat Dithiotheritol 1M DTT Kinesin (take out of freezer only before needed)
1. Prepare microtubule growth buffer solution in a microcentrifuge tube: Volume Reagent [Stock] [Final] 21.8µL BRB80 1µL Mg 100mM 4mM 1µL GTP 4mg/mL 0.16mg/mL 1.2µL DMSO 5% 0.0024% 25µL Final Volume
3. Prepare flow chamber – Use two strips of double sided tape on a 35x50 coverslip (on the long edges), and stick a 22x22 coverslip on top. (Results in ~100µm height, volume of ~15µL.) 4. Make BRB80T edited Volume Reagent [Stock] [Final] 45µL BRB80 5µL Taxol 1mM 100µM 50µL Final volume
2 trials 6. Make MT100 solution from the polymerized molecules as a control: edited Volume Reagent [Stock] [Final] 196µL BRB80 2µL Taxol 2µL MT Solution 200µL Final volume 10 trials Wrap in foil, expose to light as little as possible. Do not put on ice. Pipette MT10 as little as possible. This solution is good for 5 days approximately. Tubulin concentration in this solution is [Tubulin] = 32µg/mL 7. Prepare standard solutions: (You can prepare these during MT polymerization). If needed, make diluted stock ATP solution first for lower speed experiments BRB80CS Volume Reagent [Stock] [Final] 292.5µL BRB80 7.5µL Casein 20mg/mL 0.5mg/mL 300µL Final volume
Note: AF is good for approximately 2 hours from moment of preparation – better to make it again in the middle of the experiment than to make a large batch first. High density kinesin solution K2000 – wait 2 minutes before adding the kinesin: Volume Reagent [Stock] [Final] 89µL BRB80CS 10µL Kinesin 1µL ATP 100mM 1mM 100µL Final volume
Kinesin solution K1000 Volume Reagent [Stock] [Final] 50µL K2000 49µL BRB80CS 1µL ATP 100mM 1.5mM 100µL Final volume
8. Control motility assay – MT100, K1000 1. Flow in 20µL of BRB80CS, wait 5 minutes. 2. Flow in 20µL of K1000 motor solution, wait 5 minutes. 3. Flow in 20µL of MT100 motility solution, wait 5 minutes. From now on, keep flow cell covered. 4. Flow in 20µL of AF solution twice to wash excess tubulin. 5. Begin imaging immediately. 9. High-density motility assays – MT10, K1000 1. Flow in 20µL of BRB80CS, wait 5 minutes. 2. Flow in 20µL of K1000 motor solution, wait 5 minutes. 3. Flow in 20µL of MT10 motility solution, wait 5 minutes. From now on, keep flow cell covered. 4. Flow in 20µL of AF solution twice to wash excess tubulin. 5. Begin imaging immediately. 10. High-density motility assays – MT100, K2000. (If multiple trials were performed on the other assays, be sure to check the amount of BRB80CS you have left.) 1. Flow in 20µL of BRB80CS, wait 5 minutes. 2. Flow in 20µL of K2000 motor solution, wait 5 minutes. 3. Flow in 20µL of MT100 motility solution, wait 5 minutes. From now on, keep flow cell covered. 4. Flow in 20µL of AF solution twice to wash excess tubulin. 5. Begin imaging immediately. 11. High-density motility assays – MT10, K2000 1. Flow in 20µL of BRB80CS, wait 5 minutes. 2. Flow in 20µL of K2000 motor solution, wait 5 minutes. 3. Flow in 20µL of MT10 motility solution, wait 5 minutes. From now on, keep flow cell covered. 4. Flow in 20µL of AF solution twice to wash excess tubulin. 5. Begin imaging immediately
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