足迹
DNA
DNA结合蛋白
化学
分子生物学
生物
生物化学
转录因子
基因
古生物学
作者
Michael Brenowitz,Donald F. Senear,Robert E. Kingston
标识
DOI:10.1002/0471142727.mb1204s07
摘要
Deoxyribonuclease I (DNase I) protection mapping, or footprinting, is a valuable technique for locating the specific binding sites of proteins on DNA. The basis of this assay is that bound protein protects the phosphodiester backbone of DNA from DNase I-catalyzed hydrolysis. Binding sites are visualized by autoradiography of the DNA fragments that result from hydrolysis, following separation by electrophoresis on denaturing DNA sequencing gels. Footprinting has been developed further as a quantitative technique to determine separate binding curves for each individual protein-binding site on the DNA. For each binding site, the total energy of binding is determined directly from that site's binding curve. For sites that interact cooperatively, simultaneous numerical analysis of all the binding curves can be used to resolve both the intrinsic binding and cooperative components of these energies.DNase I footprint titration is described in this unit and involves (1) preparation of a singly end-labeled DNA restriction fragment, (2) equilibration of the protein with DNA, (3) exposure of the equilibrium mixture to DNase I, and (4) electrophoretic separation on gels of the denatured hydrolysis products, followed by autoradiography. A describes (1) densitometric analysis of the autoradiograms to obtain binding data and (2) numerical analysis of the binding data to yield binding curves and equilibrium constants for the interactions at each of the separate sites. An describes the qualitative use of footprinting to identify DNA-binding proteins in crude extracts.
科研通智能强力驱动
Strongly Powered by AbleSci AI