Dot assay for neomycin phosphotransferase activity in crude cell extracts

Abstract

A dot assay for determining neomycin phosphotransferase (NPT II) activity in crude cell extracts has been developed. The assay provides for the rapid screening of large numbers of cell cultures generated in gene transformation experiments using NPT II as a dominant selectable marker. Currently, the commonly used procedure for NPT II assay employs a time-consuming electrophoretic protein separation step to eliminate a positive interference resulting from putative protein kinase activities present in crude cell extracts. The dot method we have developed is based upon the ability of nitrocellulose membrane to eliminate that positive interference without a prior protein separation step. It provides a sensitive, reproducible, and significantly more convenient and rapid means of screening large numbers of cell extracts in order to distinguish cultures producing high levels of NPT II from those that do not.