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 Phillips1, Andres Hernandez de la Peña2

1 The Methodist Hospital Research Institute, Houston, Texas, 2 Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.28
DOI:  10.1002/0471142727.mb1028s94
Online Posting Date:  April, 2011
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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

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of a Simple Thermofluor Assay Experiment
  • Basic Protocol 2: Preparation of a Thermofluor Assay for Buffer Optimization
  • Alternate Protocol 1: Preparation of a 96‐Well Thermofluor Screen to Discover Additives that Increase Protein Stability
  • Basic Protocol 3: Preparation and Performance of the Thermofluor Assay on a qPCR Machine
  • Basic Protocol 4: Data Analysis of a Simple Thermofluor Assay Using ThermoQ Software
  • Basic Protocol 5: Analysis of Data from a Buffer Optimization Experiment Using ThermoQ Software
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of a Simple Thermofluor Assay Experiment

  Materials
  • Sypro Orange gel stain (Invitrogen)
  • Tris‐buffered saline (TBS), pH 7.5, or other suitable protein buffer
  • 1 to 10 mg/ml protein of interest
  • 2.2‐ml microcentrifuge tubes or 15‐ml plastic conical tubes
  • 96‐well PCR microplate (compatible with the qPCR machine)
  • Transparent microplate sealing foil (compatible with the qPCR machine)
  • Microplate‐compatible centrifuge
  • Additional reagents and equipment for determining protein concentration (unit 10.1)

Basic Protocol 2: Preparation of a Thermofluor Assay for Buffer Optimization

  Materials
  • Protein of interest (10 mg/ml)
  • Tris‐buffered saline, pH 7.5, or other suitable protein buffer
  • Sypro Orange gel stain (Invitrogen)
  • PACT Suite Crystallization Screen (Qiagen)
  • 1.7‐ml microcentrifuge tubes
  • PCR tubes or 8‐ or 12‐well PCR strip‐tubes
  • 96‐well PCR microplates compatible with the qPCR machine
  • 8‐ or 12‐channel micropipettor capable of delivering 10 µl
  • Transparent microplate sealing foil compatible with the microplates and qPCR machine
  • Microplate‐compatible centrifuge

Alternate Protocol 1: Preparation of a 96‐Well Thermofluor Screen to Discover Additives that Increase Protein Stability

  • Additive Screen HT (Hampton Research)

Basic Protocol 3: Preparation and Performance of the Thermofluor Assay on a qPCR Machine

  Materials
  • Light Cycler 480 (Roche) or ABI 7900 (Applied Biosystems) or iCycler (Bio‐Rad)
  • Prepared PCR plate (see protocol 1 or protocol 2)
  • ThermoQ software (http://jshare.johnshopkins.edu/aherna19/thermoq/)
  • Microsoft Excel

Basic Protocol 4: Data Analysis of a Simple Thermofluor Assay Using ThermoQ Software

  Materials
  • Computer with MATLAB (Mathworks) installed

Basic Protocol 5: Analysis of Data from a Buffer Optimization Experiment Using ThermoQ Software

  Materials
  • Computer with MATLAB (Mathworks, Natick, MA) installed
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Figures

Videos

Literature Cited

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Internet Resources
  http://jshare.johnshopkins.edu/aherna19/thermoq/
  The authors' Web site, which is dedicated to Thermofluor‐related methodology. The site contains how‐to instructions relating to method set up on various qPCR instruments and distributes ThermoQ software, allowing analysis of data obtained from diverse platforms.
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