Motor Polarity Using Fluorescent Microtubules
PM Buffer =
| 100 mM PIPES pH 6.9
2 mM EGTA
1 mM Mg2SO4
|
Special Equipment
| Beckman TLA ultracentrifuge or comparable |
Procedure
| 1. | Make rhodamine seeds by mixing 1 part rhodamine-tubulin + 4 parts PC-tubulin + MgCl2 to make final concentration of 4 mg/mL tubulin + 4 mM MgCl2 after glycerol and Mg·GTP are added. Centrifuge tubulin mixture at 155,000g at 2°C for 5 min in a Beckman TLA ultracentrifuge prior to adding glycerol and Mg·GTP. Withdraw supernatant and add 1.5 mM Mg·GTP + 20% (v/v) glycerol. Incubate 3.5 min at 37°C in 2.5 microliter volume to make rhodamine seeds. |
| 2. | Withdraw 1.5 microliters of rhodamine seeds into 4.5 microliters of tubulin mix pre-warmed 1 min at 37°C to make asymmetrically labeled fluorescent microtubules with a bright rhodamine seed at the minus end. Tubulin mix is 1 part NEM-tubulin + 1 part PC tubulin (4 mg/mL total tubulin concentration) + 2.8 mM Mg·GTP. Incubate 5 min at 37°C. Then add 19 microliters PM + 40 microliters taxol at 37°C. |
| 3. | Add asymmetrically labeled fluorescent microtubules and antifade to motility assay mixes. Observe under fluorescence using a SIT camera or cooled CCD camera. Record motility on Super VHS tape. |
Modified from W. Hancock, J. Howard Laboratory
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Created 3 June 1999 21:35 GMT
Modified 4 February 2000 19:45 GMT
