Protein detection and visualization
Facility/Node
Uppsala Protein Analysis Platform
Contact person
Ulf Landegren ulf.landegren@genpat.uu.se
Research group website http://www.genpat.uu.se/molme/
Summary of activity
There is a frequent need to search for proteins present at very low concentrations in biological specimens, or to identify specific variants of proteins or interactions among these. Our group has developed a highly sensitive and specific technique for protein detection – proximity ligation - that can be of help by permitting low protein levels to be detected, or for characterizing e.g. phosphorylated variants of proteins, provided that suitable binding reagents are available (mono- or polyclonal antibodies or other affinity reagents). The assays can be applied in solution phase or in situ for microscopic analyses. We are able to assist colleagues by providing advice and training or by establishing assays. We can also perform the analyses if required, but typically assays are transferred to the labs requesting help.
Technologies/Methods
The proximity ligation method involves the use of affinity probes, such as an affinity purified polyclonal antibody preparation or matched pairs of monoclonal antibodies. These reagents are equipped with short DNA strands, such that amplifiable DNA strands can be formed by DNA ligation when the antibodies bind the same target molecules or two closely interacting ones. Thereby signals can be efficiently amplified while minimizing nonspecific background. The technique can be of help, e.g. for analyses of potential biomarkers in biobanked samples, or to reveal functional interactions among pairs of proteins. Many variants of the standard assay procedure are possible, including solid phase or homogenous assay formats, multiplex detection reactions, and the measurement of interaction among different classes of biomolecules besides protein. The assay can also be of value for detecting low levels of infectious agents.
Practical information
In order to establish proximity ligation assays it is necessary that affinity reagents are available, and higher affinity is helpful to reach high detection sensitivity. For in situ assays, ola.soderberg@genpat.uu.se can provide further information, and masood.kamali@genpat.uu.se can answer questions about measurement of proteins in solution phase.
Details/theories of technology
The proximity ligation assay mechanism, and the various assay formats have been described in several publications.
1. Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Östman A, Landegren U. Sensitive protein detection via proximity-dependent DNA ligation assays. Nature Biotechnology 20: 473-477 (2002)
2. Gullberg M, Gústafsdóttir SM, Schallmeiner E, Jarvius J, Bjarnegård M, Betsholtz C, Landegren U, Fredriksson S. Cytokine detection by antibody-based proximity ligation. Proceedings of the National Academy of Sciences USA 101: 8420-8424 (2004)
3. Gustafsdottir SM, Schallmeiner E, Fredriksson S, Gullberg M, Söderberg O, Jarvius M, Jarvius J, Howell M, Landegren U. Proximity ligation assays for sensitive and specific protein analyses. Analytical Biochemistry 345: 2-9 (2005)
4. Gustafsdottir SM, Nordengrahn A, Fredriksson S, Wallgren P, Rivera E, Schallmeiner E, Merza M, Landegren U. Detection of individual microbial pathogens by proximity ligation. Clinical Chemistry 52: 1152-1160 (2006)
5. Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, Landegren U. Direct observation in situ of individual endogenous protein complexes. Nature Methods 3: 995-1000 (2006)
6. Schallmeiner E, Oksanen O, Ericsson O, Spångberg L, Pettersson K, Stenman UH, Landegren U. Sensitive protein detection via triple-binder proximity ligation assays. Nature Methods 4: 135-138 (2007)
7. Jarvius M, Paulsson J, Weibrecht I, Leuchowius KJ, Andersson AC, Wählby C, Gullberg M, Botling J, Sjöblom T, Markova B, Östman A, Landegren U, Söderberg O. In situ detection of phosphorylated PDGF receptor ß phosphorylation using a generalized proximity ligation method. Molecular and Cellular Proteomics 9: 1500-1509 (2007)
8. Fredriksson S, Dixon W, Ji H, Koong AC, Mindrinos M, Davis RW. Multiplexed protein detection by proximity ligation for cancer biomarker validation. Nature Methods 4: 327-329 (2007)
