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


A simple protein-DNA interaction analysis has been developed using both a high-affinity/high-specificity zinc finger protein and a low-specificity zinc finger protein with nonspecific DNA binding capability. The latter protein is designed to mimic background binding by proteins generated in randomized or shuffled gene libraries. In essence, DNA is immobilized onto the surface of microplate wells via streptavidin capture, and green fluorescent protein (GFP)-labeled protein is added in solution as part of a crude cell lysate or protein mixture. After incubation and washing, bound protein is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.4 nM protein. The assay format is ideally suited to investigate the interactions of DNA binding proteins from within crude cell extracts and/or mixtures of proteins that may be encountered in protein libraries generated by codon randomization or gene shuffling.

Divisions: College of Health & Life Sciences > School of Biosciences
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,binding,protein,DNA,protein libraries,codon randomization,gene shuffling,Biochemistry, Genetics and Molecular Biology(all),Clinical Biochemistry
Publication ISSN: 1940-9818
Last Modified: 24 Apr 2024 07:07
Date Deposited: 14 Apr 2010 12:16
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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
Hughes, Marcus D.
Morgan, Leonie J.
Santos, Albert F.
Hine, Anna V. (ORCID Profile 0000-0003-4065-831X)

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