Quantitation of cap Z in conventional actin preparations and methods for further purification of actin

Abstract

Gel-filtration is commonly used to remove contaminants from conventional 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 factor is Cap Z, a barbed end capping protein. We now establish that, even after gel-filtration, enough Cap Z can be present in conventionally prepared actin to affect events occurring at the barbed ends of actin filaments. We also demonstrate that the concentration of Cap Z can be reduced to more than a log below the KD for binding of Cap Z to actin by either 1) immunoabsorbtion of conventionally prepared actin with anti-Cap Z antibodies, or 2) an additional cycle of polymerization/depolymerization followed by repeat gel-filtration.