Materials
| Siliconized ultracentrifuge microfuge tubes |
| GTP-depleted microtubules |
| 6X SDS loading dye |
| 1X SDS loading dye |
| Coomassie Brilliant Blue R 250 (0.8% in 50% methanol + 10% acetic acid) |
| Destaining solution (13% methanol + 3% acetic acid) |
Procedure
| 1. | Mix buffer and taxol in a siliconized ultracentrifuge microfuge tube, then add motor and GTP-depleted microtubules using a set concentration of microtubules (e.g., 2 µM) and a range of motor concentrations (e.g., 0.5 - 10 µM). Click here for an example of a set of assays. |
| 2. | Incubate 5 min at room temperature. |
| 3. | Centrifuge at 50,000 rpm for 20 min at 22°C. During centrifugation, prepare 1 tube for each assay and add 5 µL 6X SDS loading dye to the tube. |
| 4. | Carefully remove the upper 20 µL of each supernatant and place into tube containing 6X SDS loading dye. |
| 5. | Remove remaining supernatant and discard. |
| 6. | Add 30 µL 1X SDS loading dye to pellet and resuspend by vortexing vigorously. |
| 7. | Boil samples 2 - 3 min and load equal amounts (e.g., 5 µL) onto an SDS-PA gel. Supernatants from assays containing greater than 2 µM motor can be loaded at half of the volume instead of the full volume for greater accuracy during quantitation. |
| 8. | Stain gel with Coomassie Blue for 1 hour and destain for 2 - 3 hours. |
| 9. | Scan gels into digital images using a conventional scanner (e.g., Agfa Arcus II scanner) or a dedicated gel scanner. The gel can be placed on a transparent yellow sheet for scanning to reduce contrast. |
| 10. | Quantitate the bound (pellet) and unbound (supernatant) motor for each assay using NIH Image (click here to obtain a copy). |
| 11. | Analyze the data using a program such as KaleidaGraph (Synergy Software), correcting the bound motor and total concentration of motor for the motor precipitated without added microtubules. Plot the concentration (µM) of unbound versus bound motor, and fit the datapoints with the Michaelis-Menton equation where y = bound motor and x = unbound motor in each assay: m1 = Vmax and m2 = Kd |
Notes
| 1. | Motor binding to microtubules is sensitive to ionic strength, which should be optimized to be high enough to prevent motor aggregation without preventing binding to microtubules. Forty to 50 mM NaCl + buffer gives good results for several motor proteins, including Ncd and Kar3. |
| 2. | Perform assays on mutant and wild-type motors on the same day using the same solutions and microtubles for more accurate comparisons. |
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