Identification of Novel DNA‐Binding Proteins Using DNA‐Affinity Chromatography/Pull Down

Abstract 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.