Motility using VE-DIC Microscopy
Anti-GST (glutathione S-transferase) antibody |
PB buffer = | 10 mM NaPO4 pH 7.2
1 mM EGTA
1 mM MgCl2
|
VALAP (1 Vasoline: 1 Lanolin: 1 Paraffin, heated gently until melted) |
Maxell XR-S120 Black Magnetite Super-VHS tapes or comparable |
Procedure
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. |
Notes
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. |
Modified from T. Salmon Laboratory
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Created 11 June 1999 20:00 GMT
Modified 27 March 2002 20:55 GMT