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SNAREs pp 191-198 | Cite as

Use of Microscale Thermophoresis (MST) to Measure Binding Affinities of Components of the Fusion Machinery

  • Robert P. Sparks
  • Rutilio Fratti
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

Abstract

Microscale thermophoresis is a relatively new technique used by an increasing number of academic laboratories to estimate relative binding affinities between ligand (analyte) that is titrated and a target (generally protein) that is either fluorescently labeled exogenously in the red or blue channel (labeled thermophoresis) or endogenously labeled via the presence of sufficient aromatic amino acid residues such as tryptophan (label-free thermophoresis). There are advantages and disadvantages to each technique; however, one major disadvantage of label-free thermophoresis is that protein-protein interactions cannot be measured, as generally most proteins have enough aromatic residues to generate an interfering signal. Thermophoresis can be used to determine steady-state binding affinities as between SNAREs and relevant binding partners of SNAREs and labeled thermophoresis is increasingly becoming a reliable technique to screen binding partners of fusion machinery to determine relevance in terms of direct biochemical interactions.

Key words

Phosphatidic acid Sec18 NSF Nanodisc SNARE 

Notes

Acknowledgments

This work was supported in part by NIH grant GM101132 to RAF.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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