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.
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky.
Brandon L
BL
Jutras
Ashutosh
A
Verma
Brian
B
Stevenson
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008863:Microspheres; D012492:Salts / Q000737:chemistry
D001426:Bacterial Proteins; D002021:Buffers; D004268:DNA-Binding Proteins; D012492:Salts; D004247:DNA