Immunofluorescence assay and flow-cytometry selection of bead-bound aptamers.
An immunofluorescence assay was developed to identify proteins specifically binding to oligonucleoside phosphorodithioate (ODN) aptamers from a bead-bound ODN library. Accordingly, NF-kappaB p50 protein was incubated with either bead-bound NF-kappaB 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.
Yang
Xianbin
X
Sealy Center for Structural Biology and Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77555-1157, USA.
Li
Xin
X
Prow
Tarl W
TW
Reece
Lisa M
LM
Bassett
Suzanne E
SE
Luxon
Bruce A
BA
Herzog
Norbert K
NK
Aronson
Judy
J
Shope
Robert E
RE
Leary
James F
JF
Gorenstein
David G
DG
D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D009841:Oligonucleotides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013873:Thionucleotides / Q000737:chemistry
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009841:Oligonucleotides; D013873:Thionucleotides