Quantitative DNase footprint titration: a tool for analyzing the energetics of protein-DNA interactions.
A major goal in biomedical research is to determine the mechanisms responsible for gene regulation. However, the promoters and operators that control transcription are often complex in nature, containing multiple-binding sites with which DNA-binding proteins can interact cooperatively. Quantitative DNase footprint titration is one of the few techniques capable of resolving the microscopic binding affinities responsible for the macroscopic assembly process. Here, we present a step-by-step protocol for carrying out a footprint titration experiment. We then describe how to quantify the resultant images to generate individual-site binding curves. Finally, we derive basic equations for binding at each site and present an overview of the fitting process, applying it to the anticipated results. Users should anticipate that the footprinting experiment will take 3-5 d starting from DNA template isolation to image acquisition and quantitation.
Connaghan-Jones
Keith D
KD
Department of Pharmaceutical Sciences, University of Colorado Denver, Denver, Colorado 80262, USA.
Moody
Amie D
AD
Bain
David L
DL
D004247:DNA / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics*; D008954:Models, Biological; D011485:Protein Binding; D023302:Protein Footprinting / Q000379:methods*
D004247:DNA; D003851:Deoxyribonucleases