RNA-Affinity Chromatography
RNA亲和色谱法
发布: 2013年07月05日第3卷第13期 DOI: 10.21769/BioProtoc.808 浏览次数: 15804
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The authors used this protocol in:
Jan 2013
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Abstract
RNA-affinity chromatography assays are used to identify proteins binding specific RNA sequences. These proteins represent potential factors contributing to the function of RNA molecules. In our lab, we have used this protocol to identify proteins binding sequence motifs involved in replication and transcription of positive strand RNA viruses. The assay described in this protocol consists on the immobilization of 5’-biotinylated RNA oligonucleotides (30-40 nt) on a streptavidin-conjugated, paramagnetic solid matrix. Then, cytoplasmic protein extracts pre-cleared on the solid matrix to decrease nonspecific binding, were incubated with the immobilized RNA molecules in the presence of a nonspecific competitor. RNA-protein complexes immobilized on the paramagnetic solid matrix were isolated using a magnet and the bound proteins were separated by polyacrylamide gel electrophoresis for proteomic analysis.
Keywords: RNA-protein interactions (RNA和蛋白质的相互作用)
Biotinylated-RNA (生物素标记的RNA)
Biotin-streptavidin interaction (生物素-相互作用)
Paramagnetic solid matrix (顺磁性固体基质)
Materials and Reagents
- 5’-biotinylated RNAs (Sigma-Aldrich)
- Streptavidin conjugated solid matrix (Dynabeads M-280 Streptavidin) (Life Technologies, InvitrogenTM, catalog number: 11205D )
- Protease inhibitor (Complete Protease Inhibitor Cocktail Tablets) (Roche, catalog number: 1697498 )
- 10% glycerol
- Non-specific competitor tRNA (Baker yeast) (Sigma-Aldrich, catalog number: R8759 )
- NuPAGE LDS sample buffer (Life Technologies, InvitrogenTM)
- Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
- Bis-Tris-Gel (Life Technologies, InvitrogenTM)
- NuPAGE MOPS SDS Running Buffer (Life Technologies, InvitrogenTM)
- Coomassie Simply Blue Safe Stain (Life Technologies, InvitrogenTM, catalog number: LC6060 )
- Diethylpyrocarbonate (DEPC) treated water
- RNase inhibitor (RNasin) (Promega, catalog number: N2611 )
- IGEPAL CA-630 (NP-40 substitute) (Sigma-Aldrich, catalog number: I3021 )
- KCl
- Glycerol
- NP-40
- EDTA
- Extraction buffer (see Recipes)
- H-BW solution (see Recipes)
- BW solution (see Recipes)
Equipment
- 150 mm plates
- Protein gel cassettes
- Orbital Shaker (J.P. SELECTA, catalog number: Orbit 3000445 )
- Magnetic particle concentrator for microcentrifuge tubes (DYNAL BIOTECH, Dynal MPC-S 120.20)
Software
- Image Lab V3.0 (Bio-Rad)
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Morales, L., Mateos-Gomez, P. A., Enjuanes, L. and Sola, I. (2013). RNA-Affinity Chromatography. Bio-protocol 3(13): e808. DOI: 10.21769/BioProtoc.808.
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分类
微生物学 > 微生物生物化学 > RNA
生物化学 > RNA > RNA-蛋白质相互作用
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