For my in vitro binding experiments with G -actin , I need to prevent spontaneous F-actin formation and maintain G-form. Please suggest how I can do it.
Hi Yesheveer,
Maybe you could add cytochalasind (D CytoD) to your reaction buffer to prevent actin polymerization. You would have to titrate the right dose for your specific experiment. CytoD blocks barbed ends, and thus actin polymerization, while - at least to my knowlege- having little effect in soluble G actin.
Hope it helps,
Cheers
@Sebastian Dupraz
Thanks for the suggestion. I'm afraid that CytoD could interfere with the binding of my test protein. I want your opinion about this?
Hi Yesheveer,
The better described mechanism for cytoD action is it capping activity.
There are also evidences it can inhibit cofilin-G-actin binding:Article Cytochalasin D acts as an inhibitor of the actin-cofilin interaction
I don't know the type of assay you are planning, but you may check the literature or maybe you have a way to perform a quick test.
Cheers,
I would like to suggest a simple way, similar to the one I use to maintain my purified G-actin stocks. I keep G-actin always in the following buffer (10mM Tris pH 7.5, 0.5mM DTT, 0.2mM ATP, 0.1mM CaCl2, 0.1mM NaN3). To prevent the formation of F-actin, the buffer should be devoid of any KCl or MgCl2 (basically any salt, since salt initiates polymerisaton). Further, before carrying out any experiment, ultracentrifuging the G-actin at high speed removes any pre-formed F-actin (during storage or otherwise).
Thank you @Pragyan Parmita Rath for the valuable suggestion. I would try that.
Always better to start with newly thawed fresh actin, as monomeric actins can't be stored well. If you need to store and reuse an aliquot, use within a week or two. Basic storage buffer (G-buffer) that I used contains 5 mM Tris–HCl (pH 8.0), 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT. But it is also recommended to supply additional ATP because of easily degradation. Keep in mind that storage temperature and actin concentration are also critical factors affecting self-polymerization. As Pragyan mentioned, high-speed centrifuge fully helps to remove actin oligomers.
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