Title | Fluorescence recovery assay for the detection of protein-DNA binding. |
Publication Type | Journal Article |
Year of Publication | 2008 |
Authors | Xu X, Zhao Z, Qin L, Wei W, Levine JE, Mirkin CA |
Journal | Anal Chem |
Volume | 80 |
Issue | 14 |
Pagination | 5616-21 |
Date Published | 2008 Jul 15 |
ISSN | 1520-6882 |
Keywords | DNA, Estrogen Receptor alpha, Fluorescence, Luminescent Measurements, Protein Binding, Receptors, Calcitriol |
Abstract | We report a novel and straightforward fluorescence recovery assay which enables the detection of protein-DNA interactions and simultaneously determines relative binding affinities of sequence-specific DNA-binding proteins for a variety of DNA sequences in a multiplexed format. The detection of protein-DNA binding is accomplished by monitoring fluorescence recovery during exonuclease digestion of DNA sequences that are modified with fluorophore-quencher pairs. Retardation of fluorescence recovery occurs with binding of the protein to the putative DNA binding element, which arrests exonuclease digestion. The assay detects protein-DNA binding in a homogeneous solution simply, quickly, and reliably without using radioisotopes. Multiplexing is possible by labeling different DNA sequences with spectrally distinct dyes, allowing simultaneous analysis of experimental and control binding reactions in the same mixture. |
DOI | 10.1021/ac8007016 |
Alternate Journal | Anal Chem |
PubMed ID | 18498181 |
PubMed Central ID | PMC8192077 |
Grant List | R01 HD20677 / HD / NICHD NIH HHS / United States U54 HD041859 / HD / NICHD NIH HHS / United States U54 CA119341 / CA / NCI NIH HHS / United States DP1 OD000285 / OD / NIH HHS / United States R01 HD020677 / HD / NICHD NIH HHS / United States |
Related Faculty:
Zhen Zhao, Ph.D.