The Combined Use of the Thermofluor Assay and ThermoQ Analytical Software for the Determination of Protein Stability and Buffer Optimization as an Aid

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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

Figure 10.28.1 A simple Thermofluor assay comparing three homologous proteins. Only the human protein exhibits a satisfactory melting curve featuring low initial fluorescence and a sigmoidal shape indicative of a protein unfolding transition. It may be of interest to protein crystallographers that only the human protein yielded diffraction‐quality crystals.

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Figure 10.28.2 Figure 1^TQ showing raw fluorescence traces obtained in each of the 96 wells of the qPCR microplate during the Thermofluor assay.

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Figure 10.28.3 Figure 2^TQ showing smoothed fluorescence traces as in Figure 1^TQ .

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Figure 10.28.4 Figure 3^TQ showing the derivative of the melting traces. The maximum value represents the midpoint of the unfolding transition, or T_m . Curves shifted the furthest to the right represent conditions in which the protein is the most stable. Ideal traces in this figure will have a symmetric, sharp, and rightward‐shifted peak.

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Figure 10.28.5 Figure 4^TQ showing the overview of the software‐determined T_m values for all wells of a 96‐well microplate. Wells with the highest T_m values feature the hottest (deep red) colors, while wells with the lowest T_m s have the coolest (deep blue) colors.

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Figure 10.28.6 Layout of the PACT screen and sample data. The PACT screen is a 96‐condition grid‐based screen, which is laid out in such a way as to allow the systematic evaluation of pH, cations, and anions. When used in conjunction with the Thermofluor assay, it allows rapid evaluation of the effects of these conditions on protein stability. The upper left quadrant of the PACT screen uses four different 3‐component buffer systems spanning pH 4 to 9. In this example, it appears that the protein is most stable between pH 6 and 7. This is apparent because for each of the four buffer systems, the T_m is highest at either pH 6 or 7. Conversely, the stability of the protein is considerably decreased when exposed to pHs 4, 5, and 9, and these conditions should be avoided. The bottom half of the PACT screen allows determination of the effect of anionic salts on protein stability. In this example none of the anionic salts assayed appear to have a large impact on stability. The upper right quadrant of the PACT screen features six common cationic salts and metals. For the protein in this example, it is apparent that the addition of calcium causes a large increase in protein stability. Calcium is a necessary cofactor for this protein and its addition should be considered when performing functional assays or crystallization trials. For specific details regarding conditions within the PACT screen, please refer to Qiagen's Web site (http://www.qiagen.com/products/protein/crystallization/compositiontables/pdf/1057791_ps_xtal_pact‐suite_update_160609_lowres.pdf).

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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.