User:Henry Hess/Notebook/Kinesin Kings/2012/06/04

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June 4, 2012: High Density Motility Assay

  • Goal: to determine whether changes in the density of microtubules and kinesin in a motility assay would yield unusual patterns.
  • Materials:

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)

  • Procedure:

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


2. Add 6.25µL of growth solution to the aliquot of tubulin (prepare microtubules in the original aliquot of tubulin). Wrap in foil, expose this to light as little as possible. This results in a final tubulin concentration of [Tubulin] = 3.2mg/mL. Incubate this on ice for 5 minutes and then incubate at 37ºC for 30 minutes. (If incubating in water bath, wrap in parafilm.)

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


5. Make MT10 solution from the polymerized microtubules: edited Volume Reagent [Stock] [Final] 32µL BRB80 4µL BRB80T 4µL MT Solution 40µL 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


BRB80CT Volume Reagent [Stock] [Final] 196µL BRB80 2µL Taxol 1mM 10µM 2µL Casein 20mg/mL 0.2mg/mL 200µL Final volume




Motility Solution (keep in dark/wrap in foil): Volume Reagent [Stock] [Final] 75µL BRB80CT 1µL DG 2M 200mM 1µL GO 20mg/mL 0.2mg/mL 1µL ATP 100mM 1mM 1µL Cat 0.8mg/mL 0.08mg/mL 1µL DTT 1M 100mM 20µL MT100 100µL Final volume


Antifade solution: Volume Reagent [Stock] [Final] 95µL BRB80CT 1µL DG 2M 200mM 1µL GO 20mg/mL 0.2mg/mL 1µL ATP 100mM 1mM 1µL Cat 0.8mg/mL 0.08mg/mL 1µL DTT 1M 100mM 100µL 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

  • Results:
    • MT100, K1000: yielded normal results. This was to be expected as this was the control group.
    • MT100, K2000:

File:20120605 1 MT100K2000 500ms.jpg

    • MT10, K1000:
    • MT10, K2000: