Skip to main content
SpringerLink
Log in

Analysis of Protein Interactions in situ by Proximity Ligation Assays

  • Chapter
  • First Online:
High-Dimensional Single Cell Analysis

Part of the book series: Current Topics in Microbiology and Immunology ((CT MICROBIOLOGY,volume 377))

Abstract

The fate of the cell is governed by interactions among proteins, nucleic acids, and other biomolecules. It is vital to look at these interactions in a cellular environment if we want to increase our understanding of cellular processes. Herein we will describe how the in situ proximity ligation assay (in situ PLA) can be used to visualize protein interactions in fixed cells and tissues. In situ PLA is a novel technique that uses DNA, together with DNA modifying processes such as ligation, cleavage, and polymerization, as tools to create surrogate markers for protein interactions of interest. Different in situ PLA designs make it possible not only to detect protein–protein interactions but also post-translational modifications and interactions of proteins with nucleic acids. Flexibility in DNA probe design and the multitude of different DNA modifying enzymes provide the basis for modifications of the method to make it suitable to use in many applications. Furthermore, examples of how in situ PLA can be combined with other methods for a comprehensive view of the cellular activity status are discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Aubele M, Spears M, Ludyga N, Braselmann H, Feuchtinger A, Taylor KJ, Lindner K, Auer G, Stering K, Hofler H, Schmitt M, Bartlett JM (2010) In situ quantification of HER2-protein tyrosine kinase 6 (PTK6) protein–protein complexes in paraffin sections from breast cancer tissues. Br J Cancer 103:663–667

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Baan B, Pardali E, ten Dijke P, van Dam H (2010) In situ proximity ligation detection of c-Jun/AP-1 dimers reveals increased levels of c-Jun/Fra1 complexes in aggressive breast cancer cell lines in vitro and in vivo. Mol Cell Proteomics 9:1982–1990

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Burke DH, Gold L (1997) RNA aptamers to the adenosine moiety of S-adenosyl methionine: structural inferences from variations on a theme and the reproducibility of SELEX. Nucleic Acids Res 25:2020–2024

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH, Lindquist RA, Moffat J, Golland P, Sabatini DM (2006) Cell profiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7:R100

    Article  PubMed Central  PubMed  Google Scholar 

  • Chen TC, Liu YW, Huang YH, Yeh YC, Chou TY, Wu YC, Wu CC, Chen YR, Cheng HC, Lu PJ, Lai JM, Huang CY (2013) Protein phosphorylation profiling using an in situ proximity ligation assay: phosphorylation of AURKA-elicited EGFR-Thr654 and EGFR-Ser1046 in lung cancer cells. PLoS One 8:e55657

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Clausson CM, Allalou A, Weibrecht I, Mahmoudi S, Farnebo M, Landegren U, Wahlby C, Soderberg O (2011) Increasing the dynamic range of in situ PLA. Nat Methods 8:892–893

    Article  CAS  PubMed  Google Scholar 

  • Conze T, Carvalho AS, Landegren U, Almeida R, Reis CA, David L, Soderberg O (2010) MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas. Glycobiology 20:199–206

    Article  CAS  PubMed  Google Scholar 

  • Coons AH, Creech HJ, Jones RN, Berliner E (1942) The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J Immunol 45:159–170

    CAS  Google Scholar 

  • Figueiredo J, Soderberg O, Simoes-Correia J, Grannas K, Suriano G, Seruca R (2013) The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC. Eur J Hum Genet 21:301–309

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gustafsdottir SM, Ostman A, Landegren U (2002) Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol 20:473–477

    Article  CAS  PubMed  Google Scholar 

  • Gajadhar A, Guha A (2010) A proximity ligation assay using transiently transfected, epitope-tagged proteins: application for in situ detection of dimerized receptor tyrosine kinases. Biotechniques 48:145–152

    Article  CAS  PubMed  Google Scholar 

  • Gajadhar AS, Bogdanovic E, Munoz DM, Guha A (2012) In situ analysis of mutant EGFRs prevalent in glioblastoma multiforme reveals aberrant dimerization, activation, and differential response to anti-EGFR targeted therapy. Mol Cancer Res 10:428–440

    Article  CAS  PubMed  Google Scholar 

  • Gomez D, Shankman LS, Nguyen AT, Owens GK (2013) Detection of histone modifications at specific gene loci in single cells in histological sections. Nat Methods 10:171–177

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Goransson J, Wahlby C, Isaksson M, Howell WM, Jarvius J, Nilsson M (2009) A single molecule array for digital targeted molecular analyses. Nucleic Acids Res 37:e7

    Article  PubMed Central  PubMed  Google Scholar 

  • Gu GJ, Friedman M, Jost C, Johnsson K, Kamali-Moghaddam M, Pluckthun A, Landegren U, Soderberg O (2013) Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation. N Biotechnol 30:144–152

    Article  CAS  PubMed  Google Scholar 

  • Gullberg M, Gustafsdottir SM, Schallmeiner E, Jarvius J, Bjarnegard M, Betsholtz C, Landegren U, Fredriksson S (2004) Cytokine detection by antibody-based proximity ligation. Proc Natl Acad Sci U S A 101:8420–8424

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gustafsdottir SM, Schlingemann J, Rada-Iglesias A, Schallmeiner E, Kamali-Moghaddam M, Wadelius C, Landegren U (2007) In vitro analysis of DNA-protein interactions by proximity ligation. Proc Natl Acad Sci U S A 104:3067–3072

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Jarvius M, Paulsson J, Weibrecht I, Leuchowius KJ, Andersson AC, Wahlby C, Gullberg M, Botling J, Sjoblom T, Markova B, Ostman A, Landegren U, Soderberg O (2007) In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method. Mol Cell Proteomics 6:1500–1509

    Article  CAS  PubMed  Google Scholar 

  • Jung J, Lifland AW, Zurla C, Alonas EJ, Santangelo PJ (2013). Quantifying RNA-protein interactions in situ using modified-MTRIPs and proximity ligation. Nucleic Acids Res 41: e12

    Google Scholar 

  • Koos B, Paulsson J, Jarvius M, Sanchez BC, Wrede B, Mertsch S, Jeibmann A, Kruse A, Peters O, Wolff JE, Galla HJ, Soderberg O, Paulus W, Ostman A, Hasselblatt M (2009) Platelet-derived growth factor receptor expression and activation in choroid plexus tumors. Am J Pathol 175:1631–1637

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Larsson C, Grundberg I, Soderberg O, Nilsson M (2010) In situ detection and genotyping of individual mRNA molecules. Nat Methods 7:395–397

    Article  CAS  PubMed  Google Scholar 

  • Larsson C, Koch J, Nygren A, Janssen G, Raap AK, Landegren U, Nilsson M (2004) In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nat Methods 1:227–232

    Article  CAS  PubMed  Google Scholar 

  • Leuchowius KJ, Clausson CM, Grannas K, Erbilgin Y, Botling J, Zieba A, Landegren U (2013) Söderberg O ²Parallel visualization of multiple protein complexes in individual cells in tumor tissue². Mol Cell Proteomics 12(6):1563–1571

    PubMed  Google Scholar 

  • Leuchowius KJ, Jarvius M, Wickstrom M, Rickardson L, Landegren U, Larsson R, Soderberg O, Fryknas M, Jarvius J (2010) High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation. Mol Cell Proteomics 9:178–183

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Leuchowius KJ, Weibrecht I, Landegren U, Gedda L, Soderberg O (2009) Flow cytometric in situ proximity ligation analyses of protein interactions and post-translational modification of the epidermal growth factor receptor family. Cytometry A 75:833–839

    Article  PubMed  Google Scholar 

  • Leuchowius KJ, Weibrecht I, Soderberg O (2011) In situ proximity ligation assay for microscopy and flow cytometry. Curr Protoc Cytom. doi:10.1002/0471142956.cy0936s56

    PubMed  Google Scholar 

  • Liu Y, Gu J, Hagner-McWhirter A, Sathiyanarayanan P, Gullberg M, Soderberg O, Johansson J, Hammond M, Ivansson D and Landegren U (2011). Western blotting via proximity ligation for high performance protein analysis. Mol Cell Proteomics 10: O111 011031

    Google Scholar 

  • Matic I, Schimmel J, Hendriks IA, van Santen MA, van de Rijke F, van Dam H, Gnad F, Mann M, Vertegaal AC (2010) Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif. Mol Cell 39:641–652

    Article  CAS  PubMed  Google Scholar 

  • Mocanu MM, Varadi T, Szollosi J, Nagy P (2011) Comparative analysis of fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA). Proteomics 11:2063–2070

    Article  CAS  PubMed  Google Scholar 

  • Nilsson M, Malmgren H, Samiotaki M, Kwiatkowski M, Chowdhary BP, Landegren U (1994) Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265:2085–2088

    Article  CAS  PubMed  Google Scholar 

  • Paulsson J, Lindh MB, Jarvius M, Puputti M, Nister M, Nupponen NN, Paulus W, Soderberg O, Dresemann G, von Deimling A, Joensuu H, Ostman A, Hasselblatt M (2011) Prognostic but not predictive role of platelet-derived growth factor receptors in patients with recurrent glioblastoma. Int J Cancer 128:1981–1988

    Article  CAS  PubMed  Google Scholar 

  • Pinto R, Carvalho AS, Conze T, Magalhaes A, Picco G, Burchell JM, Taylor-Papadimitriou J, Reis CA, Almeida R, Mandel U, Clausen H, Soderberg O, David L (2012) Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation. J Cell Mol Med 16:1474–1484

    Article  CAS  PubMed  Google Scholar 

  • Renfrow JJ, Scheck AC, Dhawan NS, Lukac PJ, Vogel H, Chandler JP, Raizer JJ, Harsh GR, Chakravarti A, Bredel M (2011) Gene-protein correlation in single cells. Neuro Oncol 13:880–885

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ribeiro AS, Sousa B, Carreto L, Mendes N, Nobre AR, Ricardo S, Albergaria A, Cameselle-Teijeiro JF, Gerhard R, Soderberg O, Seruca R, Santos MA, Schmitt F, Paredes J (2013) P-cadherin functional role is dependent on E-cadherin cellular context: a proof of concept using the breast cancer model. J Pathol 229:705–718

    Article  CAS  PubMed  Google Scholar 

  • Schubert W, Bonnekoh B, Pommer AJ, Philipsen L, Bockelmann R, Malykh Y, Gollnick H, Friedenberger M, Bode M, Dress AW (2006) Analyzing proteome topology and function by automated multidimensional fluorescence microscopy. Nat Biotechnol 24:1270–1278

    Article  CAS  PubMed  Google Scholar 

  • Sehat B, Tofigh A, Lin Y, Trocme E, Liljedahl U, Lagergren J and Larsson O (2010). SUMOylation mediates the nuclear translocation and signaling of the IGF-1 receptor. Sci Signal 3: ra10

    Google Scholar 

  • Soderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, Landegren U (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3:995–1000

    Article  PubMed  Google Scholar 

  • Soderberg O, Leuchowius KJ, Gullberg M, Jarvius M, Weibrecht I, Larsson LG, Landegren U (2008) Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods 45:227–232

    Article  PubMed  Google Scholar 

  • Tavoosidana G, Ronquist G, Darmanis S, Yan J, Carlsson L, Wu D, Conze T, Ek P, Semjonow A, Eltze E, Larsson A, Landegren UD, Kamali-Moghaddam M (2011) Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer. Proc Natl Acad Sci U S A 108:8809–8814

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510

    Article  CAS  PubMed  Google Scholar 

  • Weibrecht I, Gavrilovic M, Lindbom L, Landegren U, Wahlby C, Soderberg O (2012) Visualising individual sequence-specific protein-DNA interactions in situ. N Biotechnol 29:589–598

    Article  CAS  PubMed  Google Scholar 

  • Weibrecht I, Grundberg I, Nilsson M, Soderberg O (2011) Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells. PLoS One 6:e20148

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Weibrecht I, Leuchowius KJ, Clausson CM, Conze T, Jarvius M, Howell WM, Kamali-Moghaddam M, Soderberg O (2010) Proximity ligation assays: a recent addition to the proteomics toolbox. Expert Rev Proteomics 7:401–409

    Article  CAS  PubMed  Google Scholar 

  • Weibrecht I, Lundin E, Kiflemariam S, Mignardi M, Grundberg I, Larsson C, Koos B, Nilsson M, Soderberg O (2013) In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay. Nat Protoc 8:355–372

    Article  CAS  PubMed  Google Scholar 

  • Zieba A, Pardali K, Soderberg O, Lindbom L, Nystrom E, Moustakas A, Heldin CH and Landegren U (2012). Intercellular variation in signaling through the TGF-beta pathway and its relation to cell density and cell cycle phase. Mol Cell Proteomics 11: M111 013482

    Google Scholar 

  • Zieba A, Wahlby C, Hjelm F, Jordan L, Berg J, Landegren U, Pardali K (2010) Bright-field microscopy visualization of proteins and protein complexes by in situ proximity ligation with peroxidase detection. Clin Chem 56:99–110

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Immunology, Genetics and Pathology Science for Life Laboratory, BMC, Uppsala University, SE-751 85, Uppsala, Sweden

    Björn Koos, Linda Andersson, Carl-Magnus Clausson, Karin Grannas, Axel Klaesson, Gaëlle Cane & Ola Söderberg

Authors
  1. Björn Koos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Linda Andersson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carl-Magnus Clausson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karin Grannas
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Axel Klaesson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gaëlle Cane
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ola Söderberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ola Söderberg .

Editor information

Editors and Affiliations

  1. Center for Clinical Sciences Research, Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, USA

    Harris G. Fienberg

  2. Center for Clinical Sciences Research, Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, USA

    Garry P. Nolan

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer-Verlag Berlin Heidelberg

About this chapter

Cite this chapter

Koos, B. et al. (2013). Analysis of Protein Interactions in situ by Proximity Ligation Assays. In: Fienberg, H., Nolan, G. (eds) High-Dimensional Single Cell Analysis. Current Topics in Microbiology and Immunology, vol 377. Springer, Berlin, Heidelberg. https://doi.org/10.1007/82_2013_334

Download citation

Publish with us

Policies and ethics

Search

Navigation