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, Biotinylated-RNA, Biotin-streptavidin interaction, Paramagnetic solid matrix
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Acknowledgments
This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-167075) and the European Community’s Seventh Framework Programme (FP7/2007-2013), under the project PoRRSCon (EC grant agreement 245141). L.M. and P.A.M.-G. received a predoctoral fellowship from the Ministry of Science and Innovation of Spain (BES-2011-043489 and BES-2008-001932, respectively). We gratefully acknowledge C. M. Sanchez, and M. Gonzalez for technical assistance.
References
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