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Avoiding false negative results in specificity analysis of protein-protein interactions
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
2011 (English)In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 24, no 1, 81-89 p.Article in journal (Refereed) Published
Abstract [en]

The competition measurement using simultaneous incubation of labeled and unlabeled Ligand is a common method to assess the specificity of a biomolecular interaction. In this paper we show that invalid assumptions about the interactions may lead to improper experimental setups which in turn can result in inaccurate conclusions about the specificity. To improve understanding of competition measurements, simulations in MATLAB as well as real-time interaction analysis using LigandTracer have been performed. We show that use of a concentration of unlabeled Ligand of at least 10 × K(D) is necessary for assay accuracy. Increasing the incubation time to assure equilibrium, adding a pre-incubation phase, and a general understanding of the reversibility of an interaction may also improve the reliability of the measurement and the conclusions drawn about specificity. These findings may lower the risk of false negative results as well as reducing the amount of reagent needed.

Place, publisher, year, edition, pages
2011. Vol. 24, no 1, 81-89 p.
Keyword [en]
specificity, affinity, kinetics, competition assay
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-147025DOI: 10.1002/jmr.1026ISI: 000289781900008PubMedID: 21194119OAI: oai:DiVA.org:uu-147025DiVA: diva2:399737
Available from: 2011-02-23 Created: 2011-02-23 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions: Resolving How the Epidermal Growth Factor Binds to Its Receptor
Open this publication in new window or tab >>Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions: Resolving How the Epidermal Growth Factor Binds to Its Receptor
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cells are complex biological units with advanced signalling systems, a dynamic capacity to adapt to its environment, and the ability to divide and grow. In fact, they are of such high level of complexity that it has deemed extremely difficult or even impossible to completely understand cells as complete units. The search for comprehending the cell has instead been divided into small, relatively isolated research fields, in which simplified models are used to explain cell biology. The result produced through these reductionistic investigations is integral for our current description of biology. However, there comes a time when it is possible to go beyond such simplifications and investigate cell biology at a higher level of complexity. That time is now.

This thesis describes the development of mathematical tools to investigate intricate biological systems, with focus on heterogeneous protein interactions. By the use of simulations, real-time measurements and kinetic fits, standard assays for specificity measurements and receptor quantification were scrutinized in order to find optimal experimental settings and reduce labour time as well as reagent cost. A novel analysis platform, called Interaction Map, was characterized and applied on several types of interactions. Interaction Map decomposes a time-resolved binding curve and presents information on the kinetics and magnitude of each interaction that contributed to the curve. This provides a greater understanding of parallel interactions involved in the same biological system, such as a cell. The heterogeneity of the epidermal growth factor receptor (EGFR) system was investigated with Interaction Map applied on data from the instrument LigandTracer, together with complementing manual assays. By further introducing disturbances to the system, such as tyrosine kinase inhibitors and variation in temperature, information was obtained about dimerization, internalization and degradation rates.

In the long term, analysis of binding kinetics and combinations of parallel interactions can improve the understanding of complex biomolecular mechanisms in cells and may explain some of the differences observed between cell lines, medical treatments and groups of patients.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 854
Keyword
Heterogeneity, Kinetics, EGFR, HER2, LigandTracer, Interaction Map, Internalization, Specificity
National Category
Medical Biotechnology Cell and Molecular Biology
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-183872 (URN)978-91-554-8570-2 (ISBN)
Public defence
2013-02-15, Rudbeck Hall, Rudbeck Laboratory, Dag Hammarskjöldsväg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-01-24 Created: 2012-11-05 Last updated: 2013-02-11Bibliographically approved

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