DNA-Mediated Detection and Profiling of Protein Complexes
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules.
Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).
Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 43 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 923
Proximity ligation assay, Protein complexes, Protein interactions, Biomarkers, Prions, Antibodies
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject Medical Science
IdentifiersURN: urn:nbn:se:uu:diva-204861ISBN: 978-91-554-8718-8OAI: oai:DiVA.org:uu-204861DiVA: diva2:640552
2013-09-27, Rudbecksalen, Rudbeck Laboratory, Uppsala, 13:00 (English)
Niemeyer, Christof, professor
Landegren, Ulf, professorKamali-Moghaddam, Masood, Dr
List of papers