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DNA-protein binding assays from a single sea urchin egg: A high-sensitivity capillary electrophoresis method

Xian, Jun and Harrington, Michael G. and Davidson, Eric H. (1996) DNA-protein binding assays from a single sea urchin egg: A high-sensitivity capillary electrophoresis method. Proceedings of the National Academy of Sciences of the United States of America, 93 (1). pp. 86-90. ISSN 0027-8424. PMCID PMC40183. http://resolver.caltech.edu/CaltechAUTHORS:20141203-134301922

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Abstract

A capillary electrophoresis method has been developed to study DNA-protein complexes by mobility-shift assay. This method is at least 100 times more sensitive than conventional gel mobility-shift procedures. Key features of the technique include the use of a neutral coated capillary, a small amount of linear polymer in the separation medium, and use of covalently dye-labeled DNA probes that can be detected with a commercially available laser-induced fluorescence monitor. The capillary method provides quantitative data in runs requiring < 20 min, from which dissociation constants are readily determined. As a test case we studied interactions of a developmentally important sea urchin embryo transcription factor, SpP3A2. As little as 2-10 x 10^6 molecules of specific SpP3A2-oligonucleotide complex were reproducibly detected, using recombinant SpP3A2, crude nuclear extract, egg lysates, and even a single sea urchin egg lysed within the capillary column.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1073/pnas.93.1.86DOIArticle
http://www.pnas.org/content/93/1/86PublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC40183/PubMed CentralArticle
ORCID:
AuthorORCID
Harrington, Michael G.0000-0002-7923-8032
Additional Information:© 1996 National Academy of Sciences. Contributed by Eric H. Davidson, September 28, 1995. We thank Chiou-Hwa Yuh, James A. Coffman, Helen Lee, and Chieh Chang for their efforts in preparation and provision of the samples and reagents that we used. This work was supported by the Beckman Institute of California Institute of Technology, by National Institutes of Health Grant HD-05753, and by a subcontract from National Science Foundation Science and Technology Grant BIR9214821. J.X. was supported by National Institutes of Health Training Grant HD-07257. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Funders:
Funding AgencyGrant Number
Caltech Beckman InstituteUNSPECIFIED
NIHHD-05753
NSFBIR9214821
NIHHD-07257
Subject Keywords:mobility-shift assay; laser-induced fluorescence; transcription factor; nuclear extract
PubMed Central ID:PMC40183
Record Number:CaltechAUTHORS:20141203-134301922
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20141203-134301922
Official Citation:Xian, J., Harrington, M. G., & Davidson, E. H. (1996). DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method. Proceedings of the National Academy of Sciences, 93(1), 86-90.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:52341
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:04 Dec 2014 17:29
Last Modified:23 Feb 2016 21:06

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