Isolation of polymerization-competent cytoplasmic actin by affinity chromatography on immobilized DNAse I using formamide as eluant.
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 degrees 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 obtained in high yield and purity (greater than 90%) and can readily polymerize to F-actin.
D000199:Actins / Q000302:isolation & purification*; D000818:Animals; D002850:Chromatography, Gel; D003850:Deoxyribonuclease I; D003851:Deoxyribonucleases; D004720:Endonucleases; D005559:Formamides; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009132:Muscles / Q000032:analysis; D011817:Rabbits
D000199:Actins; D005559:Formamides; D046911:Macromolecular Substances; D003851:Deoxyribonucleases; D004720:Endonucleases; D003850:Deoxyribonuclease I