Analysis of RNA-containing compartments by hybridization and proximity labeling in cultured human cells

Karen Yap*, Tek Hong Chung, Eugene V. Makeyev

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

This protocol describes a hybridization-proximity labeling (HyPro) approach for identification of proteins and RNAs co-localizing with a transcript of interest in genetically unperturbed cells. It outlines steps required for purification of a recombinant HyPro enzyme, hybridization of fixed and permeabilized cells with digoxigenin-labeled probes, HyPro enzyme binding, proximity biotinylation, and downstream analyses of the biotinylated products. Although the protocol is optimized for relatively abundant noncoding transcripts, recommendations are provided for improving the signal-to-noise ratio in case of scarcer RNA “baits.” For complete details on the use and execution of this protocol, please refer to Yap et al. (2021).

Original languageEnglish
Article number101139
JournalSTAR Protocols
Volume3
Issue number1
Early online date28 Jan 2022
DOIs
Publication statusPublished - 18 Mar 2022

Keywords

  • Cell Biology
  • Gene Expression
  • In Situ Hybridization
  • Mass Spectrometry
  • Microscopy
  • Molecular Biology
  • Molecular/Chemical Probes
  • Protein Biochemistry
  • Protein expression and purification

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