RNA-binding patterns in total human tissue proteins: analysis by northwestern blotting.
We have developed a reproducible Northwestern (NW) blotting method to identify general patterns of RNA-binding proteins in total human tissue homogenates and have identified some of the factors contributing to this reproducibility. Unfractionated homogenates of human brain tissue were separated by SDS-PAGE, electrotransferred wet to nitrocellulose, and probed with in-vitro-transcribed labeled RNAs. Approximately ten size classes of RNA-binding proteins were observed consistently and reproducibly. Although sequence-independent electrostatic RNA-protein interactions likely contributed to most of the binding, binding to some proteins was shown to be more dependent on protein conformation: binding was not blocked by preincubation with single- or double-stranded DNA, nor with poly(A) RNA, but preincubation with tRNA revealed a distinct subset of RNA-binding proteins. In addition, preincubation with RNA, but not DNA, revealed a previously undetected RNA-binding protein of approximately 90 kDa. The NW blotting method described here can be used to reveal tissue-specific differences in RNA-binding patterns.
Chen
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Department of Neurology, Columbia University College of Physicians and Surgeons, New York, NY 10032.
Sadlock
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Schon
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D016564:Amyloid beta-Protein Precursor / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D006801:Humans; D015151:Immunoblotting; D009419:Nerve Tissue Proteins / Q000032:analysis; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000032:analysis*; D015183:Restriction Mapping; D013702:Temporal Lobe / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
D016564:Amyloid beta-Protein Precursor; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D004247:DNA