43] Ultraviolet cross-linking of DNA binding proteins

Abstract

This chapter focuses on the ultraviolet (UV) cross-linking of DNA binding proteins. To understand the regulation of transcription, it is of great importance to identify proteins that bind to DNA on the regulatory elements of the genes of interest. In general, DNA binding and transcription factor complexes can be resolved by an electrophoretic mobility shift assay (EMSA). EMSA resolves protein complexes that bind to a radiolabeled DNA fragment or double-stranded oligonucleotide probe representing the promoter or enhancer region of a gene. Although this assay provides information on the specificity and number of various complexes formed on a given DNA sequence, it fails to give further information such as the molecular weights of proteins that are directly contacting the DNA. Knowing the size of DNA binding proteins can greatly aid in the purification and cloning of the genes encoding them. In contrast to EMSA, ultraviolet radiation can bring about the formation of a covalent linkage between DNA and contacting protein, hence knowledge of specific DNA binding proteins within a protein complex can be obtained by UV cross-linking. This chapter describes the method of UV cross-linking using ĸB enhancer binding complexes as an example.