Use of microscale thermophoresis (MST) to measure binding affinities of components of the fusion machinery

Robert P. Sparks, Rutilio Fratti

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages191-198
Number of pages8
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1860
ISSN (Print)1064-3745

Fingerprint

SNARE Proteins
Proteins
Aromatic Amino Acids
Tryptophan
Ligands

Keywords

  • NSF
  • Nanodisc
  • Phosphatidic acid
  • SNARE
  • Sec18

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Sparks, R. P., & Fratti, R. (2019). Use of microscale thermophoresis (MST) to measure binding affinities of components of the fusion machinery. In Methods in Molecular Biology (pp. 191-198). (Methods in Molecular Biology; Vol. 1860). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8760-3_11

Use of microscale thermophoresis (MST) to measure binding affinities of components of the fusion machinery. / Sparks, Robert P.; Fratti, Rutilio.

Methods in Molecular Biology. Humana Press Inc., 2019. p. 191-198 (Methods in Molecular Biology; Vol. 1860).

Research output: Chapter in Book/Report/Conference proceedingChapter

Sparks, RP & Fratti, R 2019, Use of microscale thermophoresis (MST) to measure binding affinities of components of the fusion machinery. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1860, Humana Press Inc., pp. 191-198. https://doi.org/10.1007/978-1-4939-8760-3_11
Sparks RP, Fratti R. Use of microscale thermophoresis (MST) to measure binding affinities of components of the fusion machinery. In Methods in Molecular Biology. Humana Press Inc. 2019. p. 191-198. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-8760-3_11
Sparks, Robert P. ; Fratti, Rutilio. / Use of microscale thermophoresis (MST) to measure binding affinities of components of the fusion machinery. Methods in Molecular Biology. Humana Press Inc., 2019. pp. 191-198 (Methods in Molecular Biology).
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