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Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions
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Biochemistry
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JoVE Journal Biochemistry
Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

DOI:
10.3791/56004-v

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10:52 min

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September 28, 2017

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, , , ,
1Epigenetics Institute,University of Pennsylvania Perelman School of Medicine, 2Department of Cell and Developmental Biology,University of Pennsylvania Perelman School of Medicine, 3Department of Biochemistry and Biophysics,University of Pennsylvania Perelman School of Medicine, 4Graduate Group in Biochemistry and Biophysics,University of Pennsylvania Perelman School of Medicine

Chapters

  • 00:05Title
  • 01:04Crosslink of Protein-RNA Interactions in Live Cells
  • 03:26Isolation of Nuclei
  • 04:27Lysis of Nuclei and Trypsin Digestion
  • 05:51Desalting of Peptides
  • 07:39Removal of Crosslinked RNA
  • 08:44Results: RNA-binding Protein and RNA-binding Region Identification
  • 10:04Conclusion

Summary

Automatic Translation

Automatic Translation

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Tags

Sample PreparationMass SpectrometryRNA-binding ProteinsUV Cross-linking4sU LabelingCell FractionationImmunoprecipitationProtein-RNA InteractionsEpigeneticsCell CultureMouse Embryonic Stem Cells

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