39] Purification of DNA-binding proteins by site-specific DNA affinity chromatography

Abstract

Publisher Summary This chapter describes the feasibility of using site-specific DNA affinity for the purification of specific DNA-binding proteins. A precise estimate of the quantity of retarded DNA can be achieved by excising the bands from the gel and measuring—in a scintillation counter—the level of radioactivity associated with the DNA. Alternatively, the quantitation can be performed by scanning the autoradiogram in a densitometer. The DNA affinity matrix has a high protein capacity, which is determined by the number of copies of the protein recognition sequence inserted into the plasmid. It is possible to construct a plasmid in which the protein-binding sequences comprise at least 50% of the total DNA content. Once inserted into the plasmid, followed by DNA amplification in E . coli , very large quantities of these sequences can be obtained. As a result of the tight binding, elution of the protein from the site-specific DNA-cellulose column required buffers with high ionic strength.