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General Method for Isolating Targets of RNA and DNA Binding Proteins
By Robert B. Denman, Department of
Molecular Biology, Laboratory of Molecular Neurobiology, New York State Institute for
Basic Research in Developmental Disabilities, Staten Island, NY
To isolate and characterize in vivo
targets of RNA and DNA binding proteins, our laboratory used a novel strategy in which
biotinylated protein produced using TNT®
Coupled Transcription/Translation System is bound
to a SoftLinkTM Soft Release Avidin
solid support and used to capture either mRNA (from total RNA) or DNA. Bound nucleic acids
are eluted from the solid support and then amplified by differential display RT-PCR (DDRT-PCR). Bands specific for the target protein are
reamplified and cloned into the pGEM®-T Vector for subsequent sequencing. Cloned products can then be used to
define the binding motif in the target using a sensitive biotinylated protein capture
assay.
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