We have isolated actin from chicken muscle. We want to now form a uniform meshwork of actin in vitro. For this we need to first depolymerize the actin and then repolymerize them to form the network. Could anyone please suggest me any proper protocol for doing so??
Hi Asmita, there are lots of references out there that use taxol to repolymerase actin.
https://scholar.google.com/scholar?hl=en&q=actin+repolymerization+taxol&btnG=&as_sdt=1%2C21&as_sdtp=
Initially you have to isolate G-actin (monomer) using dialysis in low ionic strength buffer containing CaCl2 and ATP. After ultracentrifugation the soluble G-actin can be polymerized into polymeric F-actin by increasing the ionic strength particularly with 2 mM MgCl2. Note that impure G-actin often contains proteins that prevent actin polymerization. Hence highly pure actin is required. A protocol for rabbit actin is listed here:
Biochemistry. 1982 Jun 22;21(13):3207-14.
Cytochalasin D and platelet gelsolin accelerate actin polymer formation. A model for regulation of the extent of actin polymer formation in vivo.
Tellam R, Frieden C.
thank u for your response. ... we have isolated actin from chicken breast muscle...we will then try to depolymerize using the suggested protocols and then go on for repolymerization...
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