What Are Thermal Shift Assays?

Thermal shift assay, also known as Differential Scanning Fluorimetry (DSF), is a fluorimetry method used to observe protein unfolding that determines the protein melting temperature (Tm), defined as the point at which 50% of the protein population is unfolded. Protein thermal shift assay systems are either dye-free, monitoring changes in intrinsic fluorescence as the samples are heated, or dye-based, monitoring differences in fluorescence between the bound and unbound states of an exogenously added dye.

Compound binding or protein-protein interactions affect the melting temperature, giving a temperature shift relative to the apoprotein (delta Tm). The magnitude of this change is related to the affinity of binding. Changes in buffer composition and pH can also affect the observed Tm, making protein thermal shift assays useful tools for optimizing assay or storage buffers to improve protein stability.

Thermal shift assays can rank compounds as a primary screening technique to discover new chemical entities or as an orthogonal technique to validate hits and rankings obtained from other biophysical screening methods, such as surface plasmon resonance (SPR) or nuclear magnetic resonance (NMR) assays.

Thermal shift assay benefits

The melting temperature determined by thermal shift assays provides a direct biophysical readout of protein stability. Assays are quick to run and use little material, allowing rapid optimization of buffer systems to enhance protein stability for screening assays, protein crystallization, and cryoEM studies, dramatically increasing the success rate in those studies.

Thermal shift assays have many advantages for hit-finding studies:

  • Suitable for hit-finding with non-enzymatic targets that require biophysical detection of chemical matter
  • Appropriate for ligand binding studies with large proteins that are difficult to analyze by methods such as SPR or isothermal titration calorimetry (ITC)
  • Detect and rank binding of compounds with high μM/low mM affinity; therefore, highly suitable for fragment screening
  • Nonspecific surface interactions generally have little impact on the observed Tm; in the early stages of drug discovery, analysis of low-affinity ligands by thermal shift assays provide cleaner, more interpretable data than equivalent assays with NMR or SPR methods
  • Reagent requirements are low, setup and collection times are relatively short, and the data output is well suited to the setup of automated workflows providing rapid readout of data
  • Dye-based thermal shift assays can also be used for ligand binding studies with dsDNA, hybrid duplexes, and structured ssRNA

You can rely on our expertise using both intrinsic fluorescence and dye-based systems to optimize your assays. If compound fluorescence interferes with detection, our specialized dye screening methods offer an alternative detection window, ensuring clearer results. We optimize assays to enhance thermal shifts and improve the signal-to-noise ratio, making it easier to detect weaker hits. Whether you're working with protein targets, antibodies, or oligonucleotides, we provide full support in assay design, data interpretation, and next-step recommendations, ensuring you get the most reliable and actionable insights from your experiments.

Thermal shift assays in drug discovery

  • Hit Finding and Initial Affinity Ranking
    • Fragment or small molecule library screening and hit identification
    • Single or two-dose high-throughput screening to identify hits
    • Fast follow-up multi-dose response studies to determine the maximum delta Tm (Bmax) and concentration of ligand that gives half the maximum delta Tm (Kd-apparent) for more accurate ranking than using the single-dose delta Tm magnitude data
    • Antibody Drug Conjugate (ADC) strategy optimization and formulation optimization
  • Orthogonal Assay Confirmation of Hits from Other Screening Systems
    • Single or two-dose screening to identify corroborated hit population
    • Multi-dose response studies to determine Bmax and Kd-apparent and provide an orthogonal compound ranking to support data from other assays
  • Competition Binding Assays
    • Dose-response analysis of compound A against a fixed concentration of B
    • Inverse dose-response with compound B against a fixed concentration of A
    • Provides evidence to support co-localization or various binding sites for different compound series
  • Protein Stability Determination and Optimization of Buffers
    • Antibody stability characterization and storage buffer development
    • Optimization of assay buffers to improve assay performance
    • Optimization of buffers for crystallization and cryoEM, increasing the likelihood of successful structure determination
    • Sparse matrix screening of buffer type, pH, and salts
    • Fine grid-screening of best sparse matrix conditions
    • Screening excipients such as sugars, PEGs, and detergents


Advantages of Thermal Shift Assays

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Versatility
Applications across hit identification, hit confirmation, and assay development

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Cost-Effective
Uses standard equipment with a thermal cycler and optical detection

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High-Throughput
Assays run on 384-well plates

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Low Protein Consumption
Compared to other biophysical assays

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Frequently Asked Questions (FAQs) About Thermal Shift Assays

  • What instruments are used at Charles River for thermal shift assays?

    QuantStudio™ 7 Flex RT-PCR 384-well and BIO-RAD CFX Opus 384-well machines are available for dye-based thermal shift assays with proteins and nucleic acid. Nanotemper Prometheus Panta machine is available for dye-free NanoDSF/thermal shift assay with proteins.

  • Do I need to provide protein for protein thermal shift studies?

    No. While we are happy to work with reagents provided by clients, we can buy commercially available protein or offer a protein production service.

  • Can thermal shift assays be used for membrane proteins/protein complexes?

    Dye-based thermal shift assays cannot generally be used with membrane proteins as the dyes bind to hydrophobic regions exposed during unfolding, and membrane proteins already have large exposed hydrophobic areas. Dye-free thermal shift assays can be used with membrane proteins so long as any detergent or other excipients do not interfere with the excitation or emission window for monitoring tryptophan fluorescence. If detergent micelles are required to maintain protein solubility, this will create a biphasic system with an unknown partitioning of any ligand of interest. This may make it challenging to rank ligands.

  • Are there any restrictions in terms of protein size with thermal shift assays?

    Yes, small proteins tend to have proportionately less buried hydrophobic surface and hence give a low signal; this gives a lower limit of 15-20 kDa. Conversely, with large proteins there may be a lot of different sites and the protein may even have loosely connected macro domains with different melting temperatures. Melt curves may therefore be complex and difficult to interpret. We have successfully worked with proteins between 20 kDa and 450 kDa.