Monitoring Actin Disassembly with Time-lapse Microscopy

Published 11/08/2006
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Biology

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Summary

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Kueh, H. Y. Monitoring Actin Disassembly with Time-lapse Microscopy. J. Vis. Exp. (1), e66, doi:10.3791/66 (2006).

Abstract

Protocol

  1. Construct a flow chamber as shown in the video.  Make sure that the parafilm seal is tight and use washed coverslips.
  2. Incubate actin-binding agent in the chamber for 5-10'.
  3. Block non-specific binding sites on the glass coverslip with a blocking protein.
  4. Polymerize actin inside the chamber by flowing in G-actin in polymerizing buffer.
  5. Washout unpolymerized actin by flowing in excess buffer.

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Comments

1 Comment

  1. What is the second pair of shorter parafilm strips for? Why do you polymerize in the chamber and how? G-actin in polymerization buffer. Is't that F-actin? What is the actin binding agent and blocking protein and their buffers and their concentrations?

    Reply
    Posted by: Anonymous
    July 28, 2009 - 2:41 PM

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