32] An agarose gel electrophoresis assay for the detection of DNA-binding activities in yeast cell extracts

Abstract

This chapter discusses the use of an agarose gel electrophoresis assay for the detection of deoxyribonucleic acid (DNA)-binding activities in yeast cell extracts. The gel electrophoresis DNA-binding assay is a simple and versatile method for the quantitative detection and analysis of specific protein–DNA interactions. Recently, the polyacrylamide gel binding assay has been used to detect specific binding proteins in crude lysates of the cells of the African green monkey, Drosophila , and Escherichia coli . The agarose gel electrophoresis DNA-binding assay described in the chapter is used to identify and partially characterize a number of activities, with different DNA-binding specificities, present in yeast cell lysates. The use of agarose gels allows whole plasmids, digested into a number of restriction fragments, to be used as substrates in the assay. Specific DNA-binding activity is observed as a change in the mobility of one specific DNA fragment containing the sequence of interest; nonspecific DNA-binding activities, observed as the altered mobility of all the plasmid fragments, can be minimized by the use of unlabeled carder DNA. Competition studies with plasmid DNA containing the specifically bound DNA fragment can also be used to identify and delimit the binding substrate. Binding to genomic DNA sequences can also be detected using an adaptation of this method—the genomic-blot binding assay. In this assay, unlabeled yeast genomic DNA is used as a substrate in the binding reactions and the specific DNA mobility shifts are detected by the hybridization of a DNA probe to the DNA in a Southern blot of the gel.