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Immunofluorescence assay and flow-cytometry selection of bead-bound aptamers

By Xianbin Yang, Xin Li, Tarl W. Prow, Lisa M. Reece, Suzanne E. Bassett, Bruce A. Luxon, Norbert K. Herzog, Judy Aronson, Robert E. Shope, James F. Leary and David G. Gorenstein

Abstract

An immunofluorescence assay was developed to identify proteins specifically binding to oligonucleoside phosphorodithioate (ODN) aptamers from a bead-bound ODN library. Accordingly, NF-κB p50 protein was incubated with either bead-bound NF-κB consensus sequence or a bead-bound ODN combinatorial library and adsorption was then assessed using a specific primary antibody and a secondary antibody conjugated with Alexa 488 fluorescent dye. This assay avoids any problems related to fluorescently labeling target proteins. The method is straightforward and readily applicable to other transcription factors and proteins, and the feasibility of its application for high-throughput screening of large aptamer bead-based libraries by flow cytometry is demonstrated

Topics: NAR Methods Online
Publisher: Oxford University Press
Year: 2003
OAI identifier: oai:pubmedcentral.nih.gov:156057
Provided by: PubMed Central
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