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An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA
JoVE Journal
Biochemistry
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JoVE Journal Biochemistry
An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

DOI:
10.3791/64923-v

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07:55 min

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February 17, 2023

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, , , ,
1Center for Human Technology,Italian Institute of Technology (IIT), 2Department of Experimental Oncology,European Institute of Oncology (IEO), 3Department of Biology and Biotechnologies ‘Charles Darwin’,Sapienza University of Rome

Chapters

  • 00:05Introduction
  • 00:42Total Protein Harvest and Protein Concentration Determination
  • 01:48Bead Preparation
  • 03:12Bead Loading and Protein Binding on Beads
  • 04:13Washing of Nonspecific Binders
  • 04:53Elution of Specific Binders
  • 05:39Results: Quantification and Validation of Protein Interactors of Specific RNA Sequences
  • 07:24Conclusion

Summary

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Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

Tags

Keywords: RNA-binding ProteinsPull-down AnalysisBiotinylated RNAProtein ComplexesHEK 293T CellsLysis BufferMagnetic BeadsYeast TRNARNA Oligonucleotide

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