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Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture
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Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

DOI:
10.3791/54207-v

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10:34 min

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July 22, 2016

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, , ,
1Cardiovascular and Metabolic Disorders Program,Duke-NUS Medical School, 2Institute of Molecular and Cell Biology, Singapore, 3Division of Bioengineering, School of Chemical & Biomedical Engineering,Nanyang Technological University

Chapters

  • 00:05Title
  • 00:35Preparation of Biotin-labeled RNA
  • 02:18Preparation of RNA-conjugated Beads
  • 04:16Preparation of Cellular Lysate from Primary Adipocytes
  • 07:00Binding and Elution of RBP
  • 08:45Results: RNA Pull-down Assay
  • 10:05Conclusion

Summary

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An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Tags

Keywords: RNA-binding ProteinsRNA Pull-downAdipocyte CultureAntigen Receptor 3-Prime Untranslated Region (AR 3-Prime UTR)Biotinylated RNAIn Vitro TranscriptionStreptavidin-coated Magnetic BeadsRNA Structure BufferRNA-protein InteractionsRNA Regulatory MechanismsLong Non-coding RNA

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