Fluorescence recovery assay for the detection of protein-DNA binding. Academic Article uri icon

Overview

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.

publication date

  • May 23, 2008

Research

keywords

  • DNA
  • Estrogen Receptor alpha
  • Luminescent Measurements
  • Receptors, Calcitriol

Identity

PubMed Central ID

  • PMC8192077

Scopus Document Identifier

  • 49049120374

Digital Object Identifier (DOI)

  • 10.1021/ac8007016

PubMed ID

  • 18498181

Additional Document Info

volume

  • 80

issue

  • 14