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A Universal Approach to Prepare Reagents for DNA-Assisted Protein Analysis
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 9, e108061- p.Article in journal (Refereed) Published
Abstract [en]

The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation and extension assays, immuno-PCR, and immuno-rolling circle amplification reactions. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. Here we describe a universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions. We applied the approach to generate high-quality probes using either antibodies or recombinant affinity reagents. The purified high-grade probes were used in proximity ligation assays in solution and in situ, demonstrating both augmented assay sensitivity and improved signal-to-noise ratios.

Place, publisher, year, edition, pages
2014. Vol. 9, no 9, e108061- p.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-236454DOI: 10.1371/journal.pone.0108061ISI: 000342921200076PubMedID: 25233463OAI: oai:DiVA.org:uu-236454DiVA: diva2:764589
Available from: 2014-11-19 Created: 2014-11-19 Last updated: 2017-12-05Bibliographically approved
In thesis
1. DNA-Assisted Immunoassays for High-Performance Protein Analyses
Open this publication in new window or tab >>DNA-Assisted Immunoassays for High-Performance Protein Analyses
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins play important roles in most cellular functions, such as, replication, transcription regulation, signal transduction, for catalyzing chemical reaction, etc. Technologies developed to identify proteins rely either on observing their own properties such as charge, size, mass to charge ratio or sequence composition; or on using affinity reagents that recognize specific protein targets. Immunoassays utilizing functionalized affinity reagents are powerful for targeted proteomics. Among them, DNA-assisted immunoassays in which affinity reagents are labeled with DNA molecules, offer some unique advantages.

In this thesis, I will present works to improve current DNA-assisted immunoassays such as proximity ligation assays (PLA), as well as to take advantage of DNA reactions to adress other problems. In paper I, a new solid support (MBC-Ts) was functionalized with antibodies and used in the solid-phase PLA for detection of VEGF. The assay using MBC-Ts was compared among the commercially available solid supports in different matrices and it was shown to exhibit enhanced limit of detection in complex matrices. In paper II, a two-step protocol was described to prepare high-quality probes used in homogeneous and in situ PLA by purifying DNA-labeled affinity reagents from unconjugated affinity reagents and excess oligonucleotides. In paper III, PLA was applied on a capillary western blotting instrument so that both the sensitivity and specificity of the original assay were improved. In paper IV, a new method was introduced to profile protein components in individual protein complexes by DNA-barcoded antibodies. This method has been used to profile protein complexes such as surface proteins on individual secreted vesicles.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 53 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1055
Keyword
proximity ligation assay, magnetic beads, conjugation, phosphorylation, affinity binder, protein complexes, recombinant binder, sensitivity, specificity
National Category
Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-236591 (URN)978-91-554-9114-7 (ISBN)
Public defence
2015-01-16, B41, Biomedicalcenter, Husargatan 3, 75018, Uppsala, 09:15 (English)
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Available from: 2014-12-19 Created: 2014-11-20 Last updated: 2015-02-03

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