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Studying the model system ERK2-mAb: determining protein stability using nanoDSF and characterizing the molecular interaction using MicroScale Thermophoresis
Poster Title: Studying the model system ERK2-mAb: determining protein stability using nanoDSF and characterizing the molecular interaction using MicroScale Thermophoresis
Submitted on 18 Jan 2016
Author(s): Lukas Kniep, Estefanía Muciño, Clemens Entzian, Dr. Thomas Schubert
Affiliations: 2bind GmbH, Josef-Engert-Str. 13, 93053, Regensburg, Germany
This poster was presented at Antibodies in Drug Discovery, Cambridge UK
Poster Views: 1,655
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Poster Information
Abstract: nanoDSF (Differential Scanning Fluorimetry) is a novel label-free method used for thermal and chemical unfolding experiments to quantify the melting temperature of proteins thus their structural stability. Based on the monitoring of intrinsic tryptophan fluorescence of proteins the technology allows for revealing unfolding transition points with highresolution without modification of the protein. MicroScale Thermophoresis (MST) is an innovative method that enables the quantitative analysis of molecular interactions in solution on the microliter scale with high sensitivity. The method is based on the movement of molecules in temperature gradients, a physical effect called Thermophoresis. Basic binding parameters such as binding affinity, stoichiometry and thermodynamics are accessible using this technology. ERK2 (MAPK1, mitogen-activated protein kinase 1) is an important player in multiple biochemical signaling pathways and a highly relevant pharmaceutical target. The ERK2-mAb model system is used to demonstrate the analytical capabilities of these two innovative technologies. Initially, the buffer conditions for optimal ERK2 stability were determined (30 tested buffers), followed by the characterization of the stability of the three antibodies in the top 10 ERK2 buffers. By this procedure, a common buffer was identified, which ensured optimal thermal stability of all proteins. Based on these findings, the affinity of the antibodies towards ERK2 was studied using MicroScale Thermophoresis. Binding affinities between 5 - 200 nM were detected in solution.Summary: The ERK2-mAb model system was used to demonstrate the analytical capabilities of the two innovative technologies: nanoDSF for the analysis of protein stability and MicroScale Thermophoresis for the study of molecular interactions. Using the nanoDSF, through a buffer screening an optimal buffer for the ERK2 protein was determined. References: Dennis Breitsprecher, Nina Schlinck, David Witte, Stefan Duhr, Philipp Baaske, and Thomas Schubert; Chapter 8, Nucleic Acid Aptamers, Springer Protocols; Methods in Molecular Biology, Humana Press, 2016

Clemens Entzian, Thomas Schubert, 2bind GmbH, Josef Engert Straße 13, 93053 Regensburg, Germany, Methods, 2015 Aug 31

Thomas Schubert 1, * and Gernot Längst 2 1 2bind GmbH, Josef Engertstraße 13, 93053 Regensburg, Germany 2 Biochemie III, Universität Regensburg, Universitätstraße 31, 93053 Regensburg, Germany AIMS Biophysics, 2015 Aug 21

Zillner K, Jerabek-Willemsen M, Duhr S, Braun D, Längst G, Baaske P. Biochemistry III, Universität Regensburg, Regensburg, Germany.
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