QUANTITATION OF CAP-Z IN CONVENTIONAL ACTIN PREPARATIONS AND METHODS FOR FURTHER PURIFICATION OF ACTIN

Gel-filtration is commonly used to remove contaminants from convention al actin prepared by the method of Spudich and Watt. It has been shown that this procedure removes the majority of a factor that reduces the low-shear viscosity of actin. We have previously reported that this f actor is Cap Z, a barbed end capping protein. We now establish that, e ven after gel-filtration, enough Cap Z can be present in conventionall y prepared actin to affect events occurring at the barbed ends of acti n filaments. We also demonstrate that the concentration of Cap Z can b e reduced to more than a log below the K-D for binding of Cap Z to act in by either 1) immunoabsorbtion of conventionally prepared actin with anti-Cap Z antibodies, or 2) an additional cycle of polymerization/de polymerization followed by repeat gel-filtration. (C) 1995 Wiley-Liss, Inc.