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In situ detection of proteins by proximity ligation
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
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Manuscript (Other academic)
URN: urn:nbn:se:uu:diva-90118OAI: oai:DiVA.org:uu-90118DiVA: diva2:162332
Available from: 2003-02-07 Created: 2003-02-07 Last updated: 2010-01-13Bibliographically approved
In thesis
1. Proximity Ligation as a Universal Protein Detection Tool
Open this publication in new window or tab >>Proximity Ligation as a Universal Protein Detection Tool
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Among the great challenges in biology are the precise quantification of specific sets of proteins and analyses of their patterns of interaction on a much larger scale than is possible today.

This thesis presents a novel protein detection technique - proximity ligation - and reports the development and application of a nucleic acid amplification technique, RCA. Proximity ligation converts information about the presence or co-localization of specific proteins to unique sets of nucleic acid sequences. For detection of target proteins or protein complexes the coincident binding by pairs or triplets of specific protein-binding reagents are required. Oligonucleotide-extensions attached to those binding reagents are joined by a DNA ligase and subsequently analyzed by standard molecular genetic techniques. The technique is shown to sensitively detect an assortment of proteins using different types of binders converted to proximity probes, including SELEX aptamers and mono- and polyclonal antibodies. I discuss factors important for using the technique to analyze many proteins simultaneously.

Quantification of target molecules requires precise amplification and detection. I show how rolling circle amplification, RCA, can be used for precise quantification of circular templates using modified molecular beacons with real-time detection. The combination of proximity-probe templated circularization and RCA results in a sensitive method with high selectivity, capable of visualizing individual immobilized proteins. This technique is used for localized detection of a set of individual proteins and protein complexes at sub-cellular resolution.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 51 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1222
Molecular medicine, Proximity ligation, proteomics, aptamer, antibody, molecular beacon, rolling circle amplification, Molekylärmedicin
National Category
Medical Genetics
Research subject
Molecular Medicine
urn:nbn:se:uu:diva-3294 (URN)91-554-5521-2 (ISBN)
Public defence
2003-02-28, The Rudbeck hall, Uppsala, 09:15
Available from: 2003-02-07 Created: 2003-02-07 Last updated: 2013-06-10Bibliographically approved

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