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 JoVE Biology

Monitoring Actin Disassembly with Time-lapse Microscopy

1

1Dept. of Systems Biology, Harvard Medical School

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    Summary

    Date Published: 11/08/2006, Issue 1; doi: 10.3791/66

    Cite this Article

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

    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

    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: AnonymousJuly 28, 2009, 2:41 PM

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