Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing

Nature Protocols volume 8, pages 1234–1248 (2013)Cite this article

Subjects

A Corrigendum to this article was published on 26 March 2015

This article has been updated

Abstract

Solid-phase proximity ligation assays share properties with the classical sandwich immunoassays for protein detection. The proteins captured via antibodies on solid supports are, however, detected not by single antibodies with detectable functions, but by pairs of antibodies with attached DNA strands. Upon recognition by these sets of three antibodies, pairs of DNA strands brought in proximity are joined by ligation. The ligated reporter DNA strands are then detected via methods such as real-time PCR or next-generation sequencing (NGS). We describe how to construct assays that can offer improved detection specificity by virtue of recognition by three antibodies, as well as enhanced sensitivity owing to reduced background and amplified detection. Finally, we also illustrate how the assays can be applied for parallel detection of proteins, taking advantage of the oligonucleotide ligation step to avoid background problems that might arise with multiplexing. The protocol for the singleplex solid-phase proximity ligation assay takes ∼5 h. The multiplex version of the assay takes 7–8 h depending on whether quantitative PCR (qPCR) or sequencing is used as the readout. The time for the sequencing-based protocol includes the library preparation but not the actual sequencing, as times may vary based on the choice of sequencing platform.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Principle of spPLA.
Figure 2: Overview of the spPLA protocol.
Figure 3: Oligonucleotide design.
Figure 4: Performance of spPLA for the detection of p53.

Similar content being viewed by others

Change history

  • 16 January 2015

     In the version of this article initially published, the PCR table at Step 34C(iii) was incorrect and the sliding splint sequence in Supplementary Table 3 was also incorrect. The errors have been corrected in the HTML and PDF versions of the article and in the revised Supplementary Table 3.

References

  1. Nong, R.Y., Gu, J., Darmanis, S., Kamali-Moghaddam, M. & Landegren, U. DNA-assisted protein detection technologies. Expert Rev. Proteomics 9, 21–32 (2012).

    Article  CAS  Google Scholar 

  2. Fredriksson, S. et al. Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 20, 473–477 (2002).

    Article  CAS  Google Scholar 

  3. Lundberg, M., Eriksson, A., Tran, B., Assarsson, E. & Fredriksson, S. Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood. Nucleic Acids Res. 39, e102 (2011).

    Article  CAS  Google Scholar 

  4. Sano, T., Smith, C.L. & Cantor, C.R. Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 258, 120–122 (1992).

    Article  CAS  Google Scholar 

  5. Schweitzer, B. et al. Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection. Proc. Natl. Acad. Sci. USA 97, 10113–10119 (2000).

    Article  CAS  Google Scholar 

  6. Soderberg, O. et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Meth. 3, 995–1000 (2006).

    Article  Google Scholar 

  7. Gullberg, M. et al. Cytokine detection by antibody-based proximity ligation. Proc. Natl. Acad. Sci. U.S.A. 101, 8420–8424 (2004).

    Article  CAS  Google Scholar 

  8. Schallmeiner, E. et al. Sensitive protein detection via triple-binder proximity ligation assays. Nat. Meth. 4, 135–137 (2007).

    Article  CAS  Google Scholar 

  9. Lundberg, M. et al. Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material. Mol. Cell Proteomics 10, M110 004978 (2011).

    Article  Google Scholar 

  10. McGregor, L.M., Gorin, D.J., Dumelin, C.E. & Liu, D.R. Interaction-dependent PCR: identification of ligand-target pairs from libraries of ligands and libraries of targets in a single solution-phase experiment. J. Am. Chem. Soc. 132, 15522–15524 (2010).

    Article  CAS  Google Scholar 

  11. Darmanis, S. et al. Sensitive plasma protein analysis by microparticle-based proximity ligation assays. Mol. Cell Proteomics 9, 327–335 (2010).

    Article  CAS  Google Scholar 

  12. Darmanis, S. et al. ProteinSeq: high-performance proteomic analyses by proximity ligation and next generation sequencing. PLoS ONE 6, e25583 (2011).

    Article  CAS  Google Scholar 

  13. Wallin, U. et al. Growth differentiation factor 15: a prognostic marker for recurrence in colorectal cancer. Br. J. Cancer 104, 1619–1627 (2011).

    Article  CAS  Google Scholar 

  14. Kamali-Moghaddam, M. et al. Sensitive detection of Aβ protofibrils by proximity ligation—relevance for Alzheimer′s disease. BMC Neurosci. 11, 124 (2010).

    Article  Google Scholar 

  15. Tavoosidana, G. et al. Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer. Proc. Natl. Acad. Sci. U.S.A. 108, 8809–8814 (2011).

    Article  CAS  Google Scholar 

  16. Goransson, J. et al. Rapid identification of bio-molecules applied for detection of biosecurity agents using rolling circle amplification. PLoS ONE 7, e31068 (2012).

    Article  Google Scholar 

  17. Hammond, M., Nong, R.Y., Ericsson, O., Pardali, K. & Landegren, U. Profiling cellular protein complexes by proximity ligation with dual tag microarray readout. PLoS ONE 7, e40405 (2012).

    Article  CAS  Google Scholar 

  18. Cheng, S. et al. Sensitive detection of small molecules by competitive immunomagnetic-proximity ligation assay. Anal. Chem. 84, 2129–2132 (2012).

    Article  CAS  Google Scholar 

  19. Anderson, N.L. & Anderson, N.G. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell Proteomics 1, 845–867 (2002).

    Article  CAS  Google Scholar 

  20. Anderson, N.L. et al. Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). J. Proteome Res. 3, 235–244 (2004).

    Article  CAS  Google Scholar 

  21. Lange, V., Picotti, P., Domon, B. & Aebersold, R. Selected reaction monitoring for quantitative proteomics: a tutorial. Mol. Syst. Biol. 4, 222 (2008).

    Article  Google Scholar 

  22. Nicol, G.R. et al. Use of an immunoaffinity-mass spectrometry-based approach for the quantification of protein biomarkers from serum samples of lung cancer patients. Mol. Cell Proteomics 7, 1974–1982 (2008).

    Article  CAS  Google Scholar 

  23. Nam, J.M., Thaxton, C.S. & Mirkin, C.A. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science 301, 1884–1886 (2003).

    Article  CAS  Google Scholar 

  24. Hyndman, D.L. & Mitsuhashi, M. PCR primer design. Methods Mol. Biol. 226, 81–88 (2003).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Our work has been funded by the Knut and Alice Wallenberg Foundation, the Swedish Research Council for Medicine (2007-2720, UL); the Swedish Foundation for Strategic Research; the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115234 (OncoTrack), resources of which are composed of financial contributions from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in-kind contribution; and by the European Community's 6th and 7th Framework Programs, including a European Research Council (ERC) senior investigator grant for high-throughput protein biomarker analysis (the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement no. 294409), as well as for coordination of the EU project DiaTools focused on minimally invasive diagnostics (the European Community's 7th Framework Program (FP7/2007-2013) under grant agreement no. 259796).

Author information

Author notes

  1. Gucci Jijuan Gu

    Present address: Present address: Department of Genetics, School of Medicine, Stanford University, Stanford, California, USA.,

Authors and Affiliations

  1. Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden

    Rachel Yuan Nong, Di Wu, Junhong Yan, Maria Hammond, Gucci Jijuan Gu, Masood Kamali-Moghaddam, Ulf Landegren & Spyros Darmanis

Authors
  1. Rachel Yuan Nong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Di Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Junhong Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maria Hammond
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gucci Jijuan Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Masood Kamali-Moghaddam
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ulf Landegren
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Spyros Darmanis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to writing the protocol and accepted the final version. Questions regarding the protocol should be addressed to U.L.

Corresponding author

Correspondence to Ulf Landegren.

Ethics declarations

Competing interests

U.L. is a founder of Olink Bioscience, which is commercializing reagents for PLA and PEA.

Supplementary information

Supplementary Table 1

List of validated antibodies. (PDF 568 kb)

Supplementary Table 2

List of different oligonucleotides used in multiplex spPLA. (PDF 497 kb)

Supplementary Table 3

List of the connector oligonucleotide and universal primers used in multiplex spPLA. (PDF 485 kb)

Supplementary Table 4

List of oligonucleotides used for sequencing library preparation in multiplex spPLA. (PDF 498 kb)

Supplementary Table 5

List of oligonucleotides used in singleplex spPLA. (PDF 489 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nong, R., Wu, D., Yan, J. et al. Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing. Nat Protoc 8, 1234–1248 (2013). https://doi.org/10.1038/nprot.2013.070

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.070

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing