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Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. Rudbeck Lab, S-75185 Uppsala, Sweden..
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab. Rudbeck Lab, S-75185 Uppsala, Sweden.;Eindhoven Univ Technol, Inst Complex Mol Syst, Dept Biomed Engn, NL-5600 MB Eindhoven, Netherlands..
Protein Simple, 3001 Orchard Pkwy, San Jose, CA 95134 USA..
Protein Simple, 3001 Orchard Pkwy, San Jose, CA 95134 USA..
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, 1490Article in journal (Refereed) Published
Abstract [en]

Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP , 2017. Vol. 7, 1490
National Category
Medical and Health Sciences
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URN: urn:nbn:se:uu:diva-323033DOI: 10.1038/s41598-017-01516-7ISI: 000400554200002PubMedID: 28473697OAI: oai:DiVA.org:uu-323033DiVA: diva2:1104860
Funder
EU, European Research Council, 294409EU, FP7, Seventh Framework Programme, 241481Swedish Research CouncilSwedish Cancer Society
Available from: 2017-06-02 Created: 2017-06-02 Last updated: 2017-06-02Bibliographically approved

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