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

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.

Publication DOI: https://doi.org/10.2144/03355st06
Divisions: College of Health & Life Sciences > Aston Pharmacy School
College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences > Chronic and Communicable Conditions
College of Health & Life Sciences
College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine
Additional Information: Creative Commons Attribution Non-Commercial No Derivatives License
Uncontrolled Keywords: protein-DNA interaction,zinc finger protein,DNA,Clinical Biochemistry,Biochemistry, Genetics and Molecular Biology(all)
Full Text Link:
Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
https://www.fut ... cbi.nlm.nih.gov (Publisher URL)
PURE Output Type: Article
Published Date: 2003-11
Authors: Zhang, Zhan-Ren
Palfrey, David
Nagel, David A.
Lambert, Peter A. (ORCID Profile 0000-0002-8243-2741)
Jessop, Robert A.
Santos, Albert F.
Hine, Anna V. (ORCID Profile 0000-0003-4065-831X)

Export / Share Citation


Statistics

Additional statistics for this record