Unit

UNIT 10.28 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

  1. Kevin Phillips1,
  2. Andres Hernandez de la Peña2

Published Online: 1 APR 2011

DOI: 10.1002/0471142727.mb1028s94

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Phillips, K. and de la Peña, A. H. 2011. 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. Current Protocols in Molecular Biology. 94:VI:10.28:10.28.1–10.28.15.

Author Information

  1. 1

    The Methodist Hospital Research Institute, Houston, Texas

  2. 2

    Johns Hopkins University, Baltimore, Maryland

Publication History

  1. Published Online: 1 APR 2011
  2. Published Print: APR 2011

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. Curr. Protoc. Mol. Biol. 94:10.28.1-10.28.15. © 2011 by John Wiley & Sons, Inc.

Keywords:

  • Thermofluor;
  • high-throughput screening;
  • crystallization;
  • differential scanning fluorescence