Parallel Protein Detection by Solid‐Phase Proximity Ligation Assay with Real‐Time PCR or Sequencing

Tonge Ebai1, Masood Kamali‐Moghaddam1, Ulf Landegren1

1 Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala Biomedical Center, Uppsala University, Uppsala
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 20.10
DOI:  10.1002/0471142727.mb2010s109
Online Posting Date:  January, 2015
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Proximity ligation assays are a group of protein detection techniques in which reagents with affinity for target proteins, typically antibodies, are coupled to short strands of DNA. DNA‐modified affinity reagents are combined in assays constructed such that the coordinated binding of individual target molecules or complexes of interacting proteins by two or more of the reagents, followed by DNA ligation and/or polymerization reactions, gives rise to amplifiable DNA reporter strands. Proximity ligation assays have been shown to exhibit excellent sensitivity in single and multiplexed protein assays for individual or interacting proteins, both in solution and in situ. This unit describes procedures for developing solid‐phase proximity ligation assays for soluble proteins using either real‐time PCR or DNA sequencing as the readout. In addition, critical steps for assay optimization are discussed. © 2015 by John Wiley & Sons, Inc.

Keywords: SP‐PLA; NGS; qPCR; PTM; PLA probes

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Solid‐Phase Proximity Ligation Assay for High‐Specificity Detection of Low‐Abundance Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Solid‐Phase Proximity Ligation Assay for High‐Specificity Detection of Low‐Abundance Proteins

  • Sulfosuccinimidyl‐4 (SMCC; Thermo Scientific, cat. no. 22322)
  • Unconjugated and/or biotinylated antibodies against target protein (see Strategic Planning):
  • Two unconjugated or biotinylated antibodies for PLA probes
  • One biotinylated antibody for protein capture
  • PBS (see recipe)
  • Dithiothreitol (DTT; Sigma‐Aldrich, cat. no. D0632)
  • Conjugation buffer (see recipe)
  • Paired probe oligonucleotides (Tables 20.10.1 and 20.10.4) with desired modification (select one):
    • 100 μM thiol‐modified oligonucleotides
    • 100 nM streptavidin‐conjugated oligonucleotides (Solulink)
  • Storage buffer (see recipe)
  • Additional oligonucleotides (Tables 20.10.1 to 20.10.3)
  • 10 mg/ml Dynabeads MyOne streptavidin T1 (Life Technologies, cat. no. 65602)
  • Wash buffer (see recipe)
  • Sample of interest (e.g., plasma, serum, or whole blood)
  • Recombinant target protein for standard curve
  • PLA buffer
  • 50 mM MgCl 2
  • 10 μM TaqMan probe (IDT): 5′‐FAM‐TGACGAACCGCTTTGCCTGA‐MBG‐3′
  • 100 mM ATP (Thermo Scientific, cat. no. R0441)
  • 25 mM dNTPs (A, C, G, U; Thermo Scientific, cat. no. V0195)
  • 5 U/μl AccuStart Taq DNA polymerase with 10× PCR buffer (Quanta Biosciences, cat. no. 95061‐05K)
  • 1 U/μl T4 DNA ligase (Thermo Scientific, cat. no. EL0016)
  • 1 U/μl uracil‐DNA glycosylase (UDG; Thermo Scientific, cat. no. EN0362)
  • 10,000× SYBR Green I nucleic acid gel stain (Life Technologies, cat. no. S‐7563)
  • Dimethyl sulfoxide (DMSO; Sigma‐Aldrich, cat. no. 41648)
  • 5 U/μl Ampligase DNA ligase and 10× reaction buffer (Epicentre Biotechnology, cat. nos. A3210K and A1905B)
  • Zeba Spin desalting columns or plates, 7‐kDa MWCO (Thermo Scientific, cat. no. 89882 for 0.5‐ml columns or 89809 for plates)
  • Slide‐A‐Lyzer Mini dialysis devices, 7‐kDa MWCO (Thermo Scientific, cat. no. 69562)
  • 1.5‐ml microcentrifuge tubes
  • DynaMag‐2 Magnet and DynaMag‐96 Side Magnet (Life Technologies, cat. nos. 12321D and 12331D)
  • Rotator (Giant Bio PTR‐30 360 degree vertical multifunctional rotator)
  • PCR microtiter plate or PCR strips
  • Self‐adhesive PCR film
  • Plate centrifuge
  • Mx3005P qPCR system (Agilent Technologies)
  • Multichannel pipettor (optional)
  • 2720 thermal cycler (Applied Bioscience, cat. no. 4359659)
  • 7900 HT fast real‐time PCR system (Applied Bioscience, cat. no. 4329001)
  • 384‐well MicroAmp optical reaction plate with bar code
  • Hydra 96 microdispenser system (Robbins Scientific)
  • GFX columns (GE Healthcare, cat. no. D0632)
  • 2% (w/v) E‐gel precast agarose gel (Life Technologies, cat. no. G402002)
  • E‐gel agarose gel electrophoresis system (Life Technologies, cat. no. G6500)
  • 50‐bp DNA size ladder
Table 0.0.1   MaterialsOligonucleotides for Singleplex SP‐PLA

Name Sequence (5′ to 3′)

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Literature Cited

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