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RNA interactome capture in yeast.

Methods (San Diego, Calif.) (2016-12-21)
Benedikt M Beckmann
ABSTRACT

RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL) and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3days to complete. Next to a comprehensive explanation of the method, we focus on our findings about the choice of crosslinking in yeast and discuss the rationale of individual steps in the protocol.

MATERIALI
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Sigma-Aldrich
Yeast Synthetic Drop-out Medium Supplements, without uracil
Sigma-Aldrich
Ribonuclease T1 from Aspergillus oryzae, ammonium sulfate suspension, 300,000-600,000 units/mg protein
Sigma-Aldrich
Ribonucleasi A, (Solution of 50% glycerol, 10mM Tris-HCL pH 8.0)
Sigma-Aldrich
4-Thiouracil, 97%