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Western Blotting via Proximity Ligation for High Performance Protein Analysis
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. 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. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2011 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, no 11, O111.011031- p.Article in journal (Refereed) Published
Abstract [en]

Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor beta by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.

Place, publisher, year, edition, pages
2011. Vol. 10, no 11, O111.011031- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-162697DOI: 10.1074/mcp.O111.011031ISI: 000296759400019OAI: oai:DiVA.org:uu-162697DiVA: diva2:462426
Available from: 2011-12-07 Created: 2011-12-05 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Proximity Ligation Assay for High Performance Protein Analysis in Medicine
Open this publication in new window or tab >>Proximity Ligation Assay for High Performance Protein Analysis in Medicine
2012 (English)Doctoral thesis, comprehensive summary (Other academic) [Artistic work]
Abstract [en]

High quality reagents are preconditions for high performance protein analyses. But despite progress in some techniques, e.g. mass spectrometry, there is still a lack of affinity-based detection techniques with enhanced precision, specificity, and sensitivity. Building on the concept of multiple affinity recognition reactions and signal amplification, a proximity ligation assay (PLA) was developed as a molecular tool for analyzing proteins and their post-translational modification and interactions. PLA enhanced the analysis of protein expression levels and post-translational modifications in western blotting (Paper I), which had elevated sensitivity and specificity, and an ability to investigate protein phosphorylation.

A general and straightforward method was established for the functionalization of affinity reagents through adding DNA strands to protein domains for protein analysis in medicine (Paper II). A method for protein domain-mediated conjugation was developed to simplify the use of recombinant affinity reagents, such as designed ankyrin repeat protein (DARPin), in DNA-mediated protein analyses.

Alzheimer’s disease (AD) is characterized by progressive cognitive decline and memory impairment, and amyloid-beta plaques and neurofibrillary tangles (NFT) in the brain are clinical hallmarks of the disease. In order to understand the mechanisms underlying the formation of NFT, in situ PLA was used to explore the role of microtubule affinity related kinase 2 (MARK2) in phosphorylating tau protein during the pathological progress of AD (Paper III). The analyses of roles of MARK proteins 1-4 in phosphorylating tau protein in cells and in post-mortem human brains were performed in Paper IV.

The focus of this thesis was the study of post-translational modifications and interactions of proteins in medicine. Procedures for high performance protein analysis in western blotting via proximity ligation were developed, and a functionalization method for recombinant affinity reagents in DNA-mediated protein analysis was established. These and other techniques were used to investigate the roles of tau-phosphorylating MARK family proteins in AD.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 801
Keyword
protein, post-translational modification, interactions, protein domain, western blotting, MARK, tau, AD
National Category
Biological Sciences
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-179827 (URN)978-91-554-8449-1 (ISBN)
Public defence
2012-10-05, Rudbeck hall, Rudbeck Laboratory, Dag Hammarskjölds väg20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-09-14 Created: 2012-08-23 Last updated: 2013-01-22Bibliographically approved

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Gu, JijuanSöderberg, OlaHammond, MariaLandegren, Ulf

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