RNA-BINDING PATTERNS IN TOTAL HUMAN TISSUE PROTEINS - ANALYSIS BY NORTHWESTERN BLOTTING

被引:10
作者
CHEN, X
SADLOCK, J
SCHON, EA
机构
[1] COLUMBIA UNIV COLL PHYS & SURG,DEPT NEUROL,630 W 168TH ST,NEW YORK,NY 10032
[2] COLUMBIA UNIV COLL PHYS & SURG,DEPT GENET & DEV,NEW YORK,NY 10032
关键词
D O I
10.1006/bbrc.1993.1178
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. © 1993 Academic Press.
引用
收藏
页码:18 / 25
页数:8
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