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Steady-State and Kinetics-Based Affinity Determination in Effector-Effector Target Interactions

  • André Reinhard
  • Thorsten NürnbergerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1578)

Abstract

Dissecting the functional basis of pathogenicity and resistance in the context of plant innate immunity benefits greatly from detailed knowledge about biomolecular interactions, as both resistance and virulence depend on specific interactions between pathogen and host biomolecules. While in vivo systems provide biological context to host-pathogen interactions, these experiments typically cannot provide quantitative biochemical characterization of biomolecular interactions. However, in many cases, the biological function does not only depend on whether an interaction occurs at all, but rather on the “intensity” of the interaction, as quantified by affinity. Specifically, microbial effector proteins may bind more than one host target to exert virulence functions, and looking at these interactions more closely than would be possible in a purely black-and-white qualitative assay (as classically based on plant or yeast systems) can reveal new insights into the evolutionary arms race between host and pathogen. Recent advances in biomolecular interaction assays that can be performed in vitro allow quantification of binding events with ever greater fidelity and application range. Here, we describe assays based on microscale thermophoresis (MST) and surface plasmon resonance (SPR). Using these technologies allows affinity determination both in steady-state and in kinetic configurations, providing two conceptually independent pathways to arrive at quantitative affinity data describing the interactions of pathogen and host biomolecules.

Key words

Protein-protein interactions Affinity Kinetics Microscale thermophoresis Surface plasmon resonance 

Notes

Acknowledgments

We thank Deutsche Forschungsgemeinschaft (SFB1101) for funding.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Center of Plant Molecular Biology (ZMBP), Eberhard-Karls-University TübingenTübingenGermany

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