Use of In Situ Proximity Ligation Assays for Systems Analysis of Signaling Pathways

Tzu‐Chi Chen1, Chi‐Ying F. Huang1

1 Institute of Biopharmaceutical Sciences, National Yang‐Ming University, Taipei, Taiwan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.18
DOI:  10.1002/cpcb.1
Online Posting Date:  June, 2016
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Abstract

Understanding signaling pathway networks via protein‐protein interactions (PPIs) at the cellular level is a significant task that has not yet been completed. Here, a systems approach that computationally infers interlinked pathways from numerous PPIs is described. The endogenous PPIs can be empirically detected using an in situ proximity ligation assay (PLA), which detects and visualizes endogenous PPIs and post‐translational modifications of proteins with a high sensitivity and specificity. This unit includes two parts: (1) conversion of gene lists into PPIs for investigation and (2) large‐scale detection and analysis of endogenous PPIs for elucidating pathway networks. © 2016 by John Wiley & Sons, Inc.

Keywords: in situ proximity ligation assay (PLA); protein‐protein interaction (PPI); signaling pathway

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generate Lists of PPIs Within Signaling Pathway Networks
  • Basic Protocol 2: Detection of PPIs via In Situ Proximity Ligation Assay (PLA)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generate Lists of PPIs Within Signaling Pathway Networks

  Materials
  • Computer with access to Internet

Basic Protocol 2: Detection of PPIs via In Situ Proximity Ligation Assay (PLA)

  Materials
  • Cultured cells
  • Phosphate‐buffered saline (PBS)
  • 3% paraformaldehyde (PFA) in PBS
  • 0.2% Triton X‐100 in PBS
  • Duolink In Situ (Sigma‐Aldrich) containing:
  • Blocking solution
  • Antibody diluent
  • PLA probe (5×) stock: secondary antibody conjugates with a PLA oligonucleotide
  • Ligation buffer: contains oligonucleotides that hybridize to the PLA probes and all components, needed for ligation except the ligase
  • Ligase (1 U/μl)
  • Amplification (5×) buffer: contains all components needed for Rolling Circle Amplification except the polymerase (oligonucleotide probes labeled with a fluorophore that hybridizes to the RCA product are also included)
  • Polymerase (10 U/μl)
  • Pair of primary antibody for a given PPI (from different hosts)
  • Wash buffer (see recipe)
  • Wash buffer plus (see recipe)
  • Mounting medium with DAPI
  • Clear nail polish
  • 8‐well chamber slides (Thermo Fisher Scientific) and coverslips (Thermo Fisher Scientific)
  • 37°C incubator
  • Humidity chamber, e.g., a box with wet paper towels at the bottom
  • Staining jars
  • Shaker
  • Fluorescent or confocal microscope
  • Image analysis software, e.g., Blob Finder V3.2 (Allalou and Wahlby, ) or Cellprofiler (Carpenter et al., )
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Figures

Videos

Literature Cited

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