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Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker
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JoVE Journal Biochimica
Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

DOI:
10.3791/58697-v

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08:12 min

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January 03, 2019

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, ,
1Integrative Program for Biological and Genome Sciences,University of North Carolina at Chapel Hill, 2Department of Biochemistry and Biophysics,University of North Carolina at Chapel Hill

Capitoli

  • 00:04Titolo
  • 00:51Substrate Preparation
  • 01:28Complex Assembly
  • 02:14Immobilization of the RNA/protein Complexes on Streptavidin Beads
  • 04:00UV-Elution
  • 05:08Sample Analysis by Silver Staining Followed by Mass Spectrometry
  • 05:59Results: Analysis of UV-eluted Complexes
  • 07:36Conclusion

Summary

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Traduzione automatica

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

Tags

Keywords: RNA Protein ComplexesSingle-step PurificationBiotinPhoto-cleavable LinkerMass SpectrometryT7 Generated RNAAdaptor OligonucleotideMouse Nuclear ExtractEDTAStreptavidin Agarose BeadsBinding BufferCentrifugationTube Rotation

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