By Shalini Sharma (auth.), Ren-Jang Lin (eds.)
Due to the very important organic value of RNA and proteins functioning jointly inside a mobile, a protocol quantity describing experimental systems to review their interactions should still discover a domestic in lots of laboratories. RNA-Protein interplay Protocols, moment Edition updates, enhances, and expands upon the preferred first variation via supplying a set of state of the art recommendations built or sophisticated some time past few years in addition to tried-and-true tools. The specialist participants discover the isolation and characterization of RNA-protein complexes, the research and size of RNA-protein interplay, and similar novel recommendations and techniques. Written within the hugely profitable Methods in Molecular Biology™ sequence layout, the chapters contain short introductions to the cloth, lists of helpful fabrics and reagents, step by step, with ease reproducible laboratory protocols, and a Notes part which highlights pointers on troubleshooting and keeping off recognized pitfalls.
Comprehensive and updated, RNA-Protein interplay Protocols, moment Edition is a perfect advisor for researchers carrying on with the learn of this all-important organic partnership.
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Additional resources for RNA-Protein Interaction Protocols
5% SDS at 65 °C and analyze them using a PhosphorImager (Fuji) and the AIDA software. 6. Alternative Elution Methods for Affinity Oligonucleotide Purified RNPs Several variations for the elution of oligonucleotide affinity-purified complexes have been described. Boiling the beads in SDS-PAGE sample buffer efficiently elutes bound proteins and RNAs under denaturing conditions. Apart from destroying the complexes, this method has the disadvantage of also efficiently eluting contaminant proteins that would not elute with mild RNP-specific elution methods.
T4 DNA ligase, supplied with commercial buffer (New England Biolabs). Competent E. coli cells DH5α or XL1-Blue. Access to an automated DNA sequencing facility. 3. Preparing Extracts for Binding 1. Transformed or transfected cell line suitable for biological system under study. 2. Appropriate growth media and incubator system for above. 3. 1% Triton X-100, 10% glycerol, Complete® protease inhibitors (Roche). 4. Acid-washed glass beads 425–600 µm (Sigma). 5. Protein content assay, Micro Bicinchoninic acid assay (Pierce).
Viral titers, infection times, and cell lines should be optimized. Good results for hTERT were also obtained in sf21, a cell line that is adapted to grow in suspension, facilitating expression at a larger scale. Affinity Purification of Telomerase 21 3. hTERT levels were estimated by quantitative Western blotting comparing the signal intensity to a known concentration of an hTERT C-terminal peptide fragment. Molecular weight differences were adjusted in the quantification. 4. In vitro transcribed and purified hTERT was quantified by measuring the OD at 260 nm and applying Beer’s law: A = εCl.
RNA-Protein Interaction Protocols by Shalini Sharma (auth.), Ren-Jang Lin (eds.)