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Nucleic Acids Research, 2003, Vol. 31, No. 10 e54
© 2003 Oxford University Press

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

Xianbin Yang1, Xin Li1, Tarl W. Prow2, Lisa M. Reece3, Suzanne E. Bassett1, Bruce A. Luxon1, Norbert K. Herzog2,4, Judy Aronson2,4, Robert E. Shope2,4, James F. Leary1,2,3 and David G. Gorenstein1

1 Sealy Center for Structural Biology and Department of Human Biological Chemistry and Genetics, 2 Department of Pathology, 3 Department of Internal Medicine and 4 WHO Collaborating Center for Tropical Diseases, The University of Texas Medical Branch, Galveston, TX 77555-1157, USA

*To whom correspondence should be addressed. Tel: +1 409 747 6800; Fax: +1 409 747 6850; Email: david{at}nmr.utmb.edu

An immunofluorescence assay was developed to identify proteins specifically binding to oligonucleoside phosphorodithioate (ODN) aptamers from a bead-bound ODN library. Accordingly, NF-{kappa}B p50 protein was incubated with either bead-bound NF-{kappa}B 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.


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