Thermal Shift Assays meet HTS to discover and characterize novel protein binders
Unlocking the Power of Thermal Shift Assays for Early-Stage Drug Discovery
Thermal-shift assay (TSA) represents a powerful tool for protein-stability analysis and for discovering and characterizing new protein binders. Understanding protein stability is crucial for drug development, protein engineering, and other biotechnology applications. Among the various methods available for assessing protein stability, TSA has emerged as an effective technique. TSA provides valuable insights into protein stability, ligand interactions, and quality control, making it indispensable for drug development and protein engineering. Here, we outline the principles, methodology, and applications of TSA, as well as our recommendations for use in early-stage drug discovery and our TSA capabilities to identify and characterize protein binders for your target protein.
What are thermal shift assays?
TSA is a highly parallelizable, cost-effective technique for studying protein stability and protein binders. It measures the thermal denaturation of a target protein by monitoring the fluorescence intensity of a dye that binds to exposed hydrophobic regions of the protein during heating. As the protein unfolds, the exposed hydrophobic regions increase, leading to an increase in fluorescence signal. Compounds, such as inhibitors or in general modulators, stabilize the three-dimensional conformation of the target protein and as a consequence the temperature needed to denature the protein is higher (see purple curve in left figure).
The figure on the right below shows fluorescence intensity curves (top panel). The blue curve is the target protein with the dye; the other traces correspond to the addition of increasing concentration of a binder compound that causes a shift of the melting temperature of the protein complex. The melting temperature can be calculated as the inflection point of the fluorescence intensities or as the first derivative of the curves (peaks in the bottom right panel).
TSA and differential scanning fluorimetry (DSF) can generally be used interchangeably, a primary distinguishing feature between the two lies in their denaturation methods. TSA specifically refers to the denaturation of a target protein triggered solely by temperature intensification. On the other hand, DSF encompasses techniques that employ various physicochemical agents, including but not limited to temperature intensification, in order to induce denaturation in the target protein. In this article, our focus will be directed towards exploring the specific aspects of TSA.
Axxam’s TSA capabilities
At Axxam, we offer a range of TSA capabilities to support your research needs:
- TSA instrument platform: our TSA experiments are conducted using advanced thermocyclers that allow for the creation of precise temperature gradients ranging from 20 to 100 °C. These instruments are equipped with the capability to accurately read the fluorescence emission of SYPROTM Orange dye, supplying reliable data for your TSA analysis.
- Screening compatibility: for projects requiring higher throughput, we have the capability to run TSA in a 384-well plate format. This screening choice allows for efficient analysis of a larger number of samples. Moreover, we have integrated suitable instruments into our automated screening station, ensuring streamlined processes and precise data acquisition.
- Production of target protein in-house: our highly experienced biochemistry unit has the expertise to produce the required quantities of your target protein. Whether you need wild-type or truncated forms, we can generate the proteins in-house, ensuring quality control and efficiency in the process.
- Proprietary fluorescent dye library: notably reported in literature, SYPROTM Orange dye works with around 70% of soluble proteins and for this reason we developed a proprietary dye library based on the binding mechanism of SYPROTM Orange but with diverse chemical scaffolds unrelated to this particular dye. Thus, if the target protein is not TSA-detectable with SYPROTM Orange, we have the possibility to screen our dye library with the aim of obtaining useful data using a different dye.
By leveraging our TSA capabilities, you can benefit from our in-house protein production, advanced instrument platform, and screening and profiling options. We are committed to providing reliable and efficient TSA services to support your research and drug discovery endeavors.