Photoactivated In Vivo Proximity Labeling

David B. Beck1, Roberto Bonasio2

1 Department of Medicine, Columbia University Medical Center, New York, New York, 2 Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.18
Online Posting Date:  June, 2017
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Abstract

Identification of molecular interactions is paramount to understanding how cells function. Most available technologies rely on co‐purification of a protein of interest and its binding partners. Therefore, they are limited in their ability to detect low‐affinity interactions and cannot be applied to proteins that localize to difficult‐to‐solubilize cellular compartments. In vivo proximity labeling (IPL) overcomes these obstacles by covalently tagging proteins and RNAs based on their proximity in vivo to a protein of interest. In IPL, a heterobifunctional probe comprising a photoactivatable moiety and biotin is recruited by a monomeric streptavidin tag fused to a protein of interest. Following UV irradiation, candidate interacting proteins and RNAs are covalently biotinylated with tight spatial and temporal control and subsequently recovered using biotin as an affinity handle. Here, we describe experimental protocols to discover novel protein‐protein and protein‐RNA interactions using IPL. © 2017 by John Wiley & Sons, Inc.

Keywords: proximity labeling; protein‐protein interactions; protein‐RNA interactions; protein complexes; ribonucleoprotein complexes; noncoding RNAs

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: In Vivo Proximity Labeling
  • Basic Protocol 2: Purification of Protein Candidates
  • Basic Protocol 3: Purification of RNA Candidates
  • Support Protocol 1: Generation and Validation of Inducible Transgenic Cell Lines for IPL
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Vivo Proximity Labeling

  Materials
  • Stable cell lines expressing the protein of interest fused to monomeric streptavidin or control tag, e.g., GFP (see Support Protocol 1)
  • T‐REx‐293 cells (Thermo Fisher, cat. no. R71007) or other expression systems can also be used, as discussed in Strategic Planning.
  • Mammalian cell culture medium (see recipe)
  • 100 mg/ml Zeocin (Invivogen, cat. no. ant‐zn‐1)
  • 0.05% trypsin (HyClone, cat. no. SH30236.02)
  • 1× PBS/2 mM EDTA (see recipe for 10×, dilute 1:10 with sterile H 2O, add EDTA to 2 mM final)
  • Doxycycline (Sigma, cat. no. D9891), stock 1 mg/ml in PBS, sterilize by filtration, store in the dark, do not freeze/thaw more than 10 times
  • 10 mM biotin‐TFPA (EZ‐Link TFPA‐PEG3‐Biotin, Pierce/Thermo Fisher, cat. no. 21303) in DMSO
  • Biotin‐TFPA is sensitive to light. Prepare single‐use aliquots, and store at −80°C in the dark.
  • Ice‐cold PBS
  • 100 mM PMSF (Sigma, cat. no. P7626) in 100% methanol (protect from light, and store at room temperature)
  • Mammalian cell culture incubator at 37°C, 5% CO 2
  • 10‐cm tissue culture‐treated culture dishes (Thermo Fisher, cat. no. 12‐565‐020)
  • UV crosslinker equipped with 365‐nm wavelength (UVA) bulbs (e.g., Spectroline Spectrolinker, cat. no. XL1500A)
  • Cell scrapers (Thermo Fisher, cat. no. 08‐100‐240)
  • 15‐ml conical tubes (Thermo Fisher, cat. no. 12565268)

Basic Protocol 2: Purification of Protein Candidates

  Materials
  • Biotinylated cell pellet ( protocol 1)
  • IPL cell lysis buffer (see recipe)
  • Streptavidin‐conjugated agarose beads (Millipore, cat. no. 69203)
  • 20% (w/v) SDS (Thermo Fisher, cat. no. AM9820)
  • IPL cell lysis buffer without detergents (see recipe)
  • Modified Laemmli's sample buffer (see recipe)
  • 200 mM biotin (Sigma, cat. no. B4501) in DMSO, store at 4°C
  • Sonicator (e.g., Model 120 Sonic Dismembrator, Fisher Scientific, cat. no. FB120A110) equipped with suitable probe (e.g., 0.3 cm probe; Fisher Scientific, cat. no. FB4422)
  • Centrifugal filters (Millipore, cat. no. UFC30SV00)

Basic Protocol 3: Purification of RNA Candidates

  Materials
  • Biotinylated cell pellet (from protocol 1)
  • TRIzol reagent (Ambion/Thermo Fisher, cat. no. 15596‐026)
  • Chloroform (Sigma, cat. no. C2432)
  • 15 mg/ml glycoblue (Ambion/Thermo Fisher, cat. no. AM9515)
  • Isopropanol (Sigma, cat. no. 278475)
  • 70% and 80% ethanol (Decon Labs, cat. no. 2701)
  • BTE buffer (see recipe)
  • TURBO DNase (Ambion/Thermo Fisher, cat. no. AM2238), includes 10× TURBO DNase Buffer
  • MyOne Streptavidin C1 Dynabeads (Thermo Fisher, cat. no. 65001)
  • RIP buffer (see recipe)
  • RIP‐W buffer (see recipe)
  • 200 mM biotin (Sigma, cat. no. B4501) in DMSO, store at 4°C
  • 1.5‐ml microcentrifuge tubes (VWR/Neptune, cat. no. 4445.S.X)
  • NanoDrop spectrophotometer (Thermo Fisher, cat. no. ND‐2000) or equivalent
  • DynaMag‐2 Magnet (Thermo Fisher, cat. no. 12321D) or equivalent
  • 1.5‐ml DNA LoBind microcentrifuge tubes (Eppendorf, cat. no. 022431021)

Support Protocol 1: Generation and Validation of Inducible Transgenic Cell Lines for IPL

  Additional Materials
  • T‐REx‐293 cells (Thermo Fisher, cat. no. R71007)
  • Plasmid encoding the protein of interest fused to monomeric streptavidin (e.g., pINTO‐NSA::mEzh2; Addgene, cat. no. 61725)
  • Plasmid encoding protein of interest fused to a control tag (e.g., pINTO‐NFH::mEzh2; Addgene, cat. no. 65925)
  • Lipofectamine 3000 Reagent (Thermo Fisher, cat. no. L3000075)
  • 1.5‐ml microcentrifuge tubes (VWR/Neptune, cat. no. 4445.S.X)
  • CHAPS/urea buffer (see recipe)
  • Bradford protein assay (Bio‐Rad, cat. no. 5000006)
  • 10‐cm tissue culture dishes, treated (Thermo Fisher, cat. no. 12‐565‐020)
  • 6‐well tissue culture plates, treated (Thermo Fisher, cat. no. 14‐832‐11)
  • 24‐well tissue culture plates, treated (Thermo Fisher, cat. no. 12‐565‐163)
  • Cloning cylinders (Sigma Aldrich, cat. no. CLS31666)
  • Sonicator (e.g., Model 120 Sonic Dismembrator, Fisher Scientific, cat. no. FB120A110) equipped with suitable probe (e.g., 0.3 cm probe, Fisher Scientific, cat. no. FB4422)
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Figures

Videos

Literature Cited

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