Fluorescent microplate-based analysis of protein-DNA interactions I: immobilized protein

Zhan-Ren Zhang, David Palfrey, David A. Nagel, Peter A. Lambert, Robert A. Jessop, Albert F. Santos, Anna V. Hine

Research output: Contribution to journalArticle

Abstract

A simple protein-DNA interaction analysis has been developed using a high-affinity/high-specificity zinc finger protein. In essence, purified protein samples are immobilized directly onto the surface of microplate wells, and fluorescently labeled DNA is added in solution. After incubation and washing, bound DNA is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.2 nM DNA. Since the detection of bound DNA is noninvasive and the protein-DNA interaction is not disrupted during detection, iterative readings may be taken from the same well, after successive alterations in interaction conditions, if required. In this respect, the assay may therefore be considered real time and permits appropriate interaction conditions to be determined quantitatively. The assay format is ideally suited to investigate the interactions of purified unlabeled DNA binding proteins in a high-throughput format.
Original languageEnglish
Pages (from-to)980-986
Number of pages7
JournalBiotechniques
Volume35
Issue number5
DOIs
Publication statusPublished - Nov 2003

Bibliographical note

Creative Commons Attribution Non-Commercial No Derivatives License

Keywords

  • protein-DNA interaction
  • zinc finger protein
  • DNA

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