| Anti-GST (glutathione S-transferase) antibody |
| PB buffer = | 10 mM NaPO4 pH 7.2 1 mM EGTA 1 mM MgCl2 |
| Clarified motor lysate |
| MTs or Axoneme-MTs |
| 50 mM Mg·ATP |
| Kimwipes |
| VALAP (1 Vasoline: 1 Lanolin: 1 Paraffin, heated gently until melted) |
| Maxell XR-S120 Black Magnetite Super-VHS tapes or comparable |
Procedure
Notes
Modified from T. Salmon Laboratory
1. Spin a clean coverslip dry using a home-made spinner.
2.
Coat with 8 microliters anti-GST antibody and tilt from side to side for 1 minute.
3.
Rinse off excess antibody from the coverslip with 2 x 30 microliters PB, spinning briefly between additions of buffer. Do not let the coverslip dry out.
4.
Add sequentially: 6.5 microliters clarified motor lysate
1.6 microliters MTs or Axnomene-MTs
0.9 microliter 50 mM Mg·ATP pH 7
9.0 microliters
5.
Place coverslip on a clean microscope slide and seal edges with VALAP.
6.
Visualize MTs or Axoneme-MTs by video-enhanced differential interference contrast (VE-DIC) microscopy. Record MT gliding onto a Super-VHS videotape.
1. Axoneme-MTs can be used in these assays instead of unlabeled MTs to determine polarity of motor movement.
2. Fluorescent MTs can be used in these assays instead of unlabeled MTs. Record motility using a SIT camera or cooled CCD camera.
3. Microscope slides are cleaned by rinsing in ethanol and wiping dry with Kimwipes.
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Created 11 June 1999 20:00 GMT
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Modified 27 March 2002 20:55 GMT