The problem with my current protocol is that the actin filaments after polymerisation are very short. I was only given with G-actin, Biotinated G-actin, Polymerisation Buffer (No ATP), General Actin Buffer and Phalloidin to start. Are there any other similar protocols? Or any suggestions? Thanks!!!!!!
I have had good results using the protocol outlined in this paper:
Huang, S., L. Blanchoin, D.R. Kovar and C.J. Staiger. 2003. Arabidopsis capping protein (AtCP) is a heterodimer that regulates assembly at the barbed ends of actin filaments. J. Biol. Chem. 278: 44832-44842. in the section Fluorescence Microscopy of Actin Filaments, in the Materials and Methods section at the end of the paper. One key tip is that you need to dilute out the filaments after polymerization to measure their length. When diluting out the filaments, they are very susceptible to shearing during pipetting. I use a pipet tip with the end cut off and the cut end edges rounded off by heating briefly in a bunsen burner. Pipet very slowly to dilute.
Good luck!
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