Motility using VE-DIC Microscopy

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

      Clarified motor lysate

      MTs or Axoneme-MTs

      50 mM Mg·ATP


      VALAP (1 Vasoline: 1 Lanolin: 1 Paraffin, heated gently until melted)

      Maxell XR-S120 Black Magnetite Super-VHS tapes or comparable


      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.

    Modified from T. Salmon Laboratory

If you use methods from the Kinesin site, we ask that you cite the Kinesin Home Page and authors, or the appropriate source publication in your work.

Copyright 1996-2003. All rights reserved.

Return to the Methods Page.

Return to the Kinesin Home Page.

Created 11 June 1999 20:00 GMT

Modified 27 March 2002 20:55 GMT