UNIT 1F.1 Identification of Novel DNA-Binding Proteins Using DNA-Affinity Chromatography/Pull Down

This units presents methods through which one may isolate and identify novel bacterial DNA-binding proteins. Briefly, the DNA sequence of interest is affixed to beads, and then incubated with bacterial cytoplasmic extract. Washes with buffers containing nonspecific DNA and low-salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute more specific DNA-binding proteins. Eluted proteins may then be identified by standard proteomic techniques. Curr. Protoc. Microbiol. 24:1F.1.1-1F.1.13. © 2012 by John Wiley & Sons, Inc.

Keywords: DNA-binding protein; prokaryote; gene regulation