Isolation of Polymerization‐Competent Cytoplasmic Actin by Affinity Chromatography on Immobilized DNAse I Using Formamide as Eluant

Author:
Open Access
Publisher website
Google Scholar
Abstract
Formamide dissociates the G‐actin · DNAse‐I complex and is therefore suitable as an alternative to the eluant containing 3 M guanidinium hydrochloride, suggested originally by Lazarides and Lindberg [Proc. Natl Acad. Sci. USA, 71, 4742–4746 (1974)], to elute actin from immobilized DNAse‐I–agarose. Formamide provides the advantage of being a much weaker denaturant than guanidinium hydrochloride and being a nonionic substance. In the concentration necessary for the dissociation of the G‐actin · DNAse‐I complex (approximately 10 M) formamide denatures actin only slowly (half‐time approximately 150 min at 2°C) and thus allows the recovery of a large fraction of actin in a polymerization‐competent form from the affinity column.Based on these findings a rapid two‐step procedure for the isolation of non‐muscle G‐actin from cultured cells is described. The actin is obtained in high yield and purity (greater than 90%) and can readily polymerize to F‐actin.

This publication has 20 references indexed in Scilit: