The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization.

Kevin Phillips, Andres Hernandez de la Peña

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

The Thermofluor assay, also referred to as a thermal shift assay or differential scanning fluorescence (DSF), is a fast and simple method that is based upon high-throughput measurements of protein stability. The Thermofluor method can be performed on nearly all qPCR machines, meaning no special instrumentation is necessary, and can be used to validate the quality of protein preparations, screen for ligands or cofactors, and discover buffers and additives that maximize protein stability. This unit describes how to set up a Thermofluor method on several common models of qPCR instruments, how to prepare the samples for the assay, and how to run and analyze the resulting data. The unit also describes a 96-well screen to determine optimal buffer conditions for protein stability, which may assist in protein crystallization, and details the use of custom software, developed specifically for the analysis of data from Thermofluor screens.

Original languageEnglish
JournalCurrent Protocols in Molecular Biology
VolumeChapter 10
Issue numberSUPPL.94
DOIs
StatePublished - Apr 1 2011
Externally publishedYes

Keywords

  • Crystallization
  • Differential scanning fluorescence
  • High-throughput screening
  • Thermofluor

ASJC Scopus subject areas

  • Molecular Biology

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