Identification of Previously Unknown DNA‐Binding Proteins Using DNA Affinity/Pull‐Down Methods Followed by Mass Spectrometry

This protocol presents methods for isolating and identifying novel nucleic acid–binding proteins. We focus on bacterial DNA‐binding proteins, although the methods can be readily adapted to identify nucleic acid–binding proteins of eukaryotes or archaea, and proteins that bind to RNAs. 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 DNA‐binding proteins that have greater specificity. Eluted proteins are then identified by standard proteomic techniques, such as mass spectrometry.