Applications of fluorescence anisotropy to the study of protein-DNA interactions.
The use of fluorescence anisotropy to monitor protein-DNA interactions has been on the rise since its introduction by Heyduk and Lee in 1990. As a solution-based, true-equilibrium, real-time method, it has several advantages (and a few disadvantages) relative to the more classical methods of filter binding and the electrophoretic mobility shift assay (gel shift). This chapter discusses the basis for monitoring protein-DNA interactions using fluorescence anisotropy, as well as the advantages and disadvantages of the method, but the bulk of the chapter is devoted to experimental tips and guidance meant to augment existing reviews of the method. The focus is on the current primary use of the method: direct measurement of binding isotherms for protein-DNA interactions in vitro. A short summary of emerging applications of the method is also included.
LiCata
Vince J
VJ
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
Wowor
Andy J
AJ
D002138:Calibration; D004247:DNA / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004058:Diffusion; D004492:Edetic Acid; D005454:Fluorescence Polarization / Q000295:instrumentation* / Q000379:methods*; D007700:Kinetics; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism; D012399:Rotation; D013696:Temperature
D011506:Proteins; D012235:Rhodamines; D004247:DNA; D004492:Edetic Acid; D004259:DNA-Directed DNA Polymerase