Fluorescence-based Thermal Shift Assay

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Fluorescence-based thermal shift assay (FTSA) is well-regarded as a frontline primary screening assay that can be used to quickly identify hits against a target or discover buffer and pH preferences of your protein.

Jumpstart any drug discovery project by quickly assessing the druggability of your target and finding the ideal buffer for ongoing experiments using FTSA. 

A typical application for FTSA is to measure the ability of a compound to thermally stabilize a protein – the assumption being that binding of the compound will increase the melting temperature of the protein. The same concept can be applied to protein-protein interactions including antibodies.

Under certain conditions, FTSA can also be used to measure the affinity between compounds and protein – crucial data for any drug discovery project.

At Charnwood Discovery we use FTSA to complement and validate orthogonal biophysical techniques, such as SPR. The benefits of using FTSA for your project include:

  • Potentially low protein consumption – depending on the target protein FTSA can be used with very low amounts of recombinant protein, allowing us to work with difficult to purify targets
  • Extremely high throughput – FTSA is a very fast technique allowing us to screen thousands of compounds per day – allowing a rapid start to any project
  • Inexpensive to run – without the need for costly reagents or consumables FTSA helps maximize the efficient use of a project budget
  • High chances of success – the chances of successful assay development for FTSA are generally high – it is applicable to the majority of folded, globular proteins
FTSA

In figure 1 we see the raw, replicate melt curves for the thermal unfolding of BRD4.

Typically we exclude the data either side of the main transition (figure 2) to leave us a with a single, smooth sigmoidal curve – fitting this with an appropriate model gives us the protein’s melting temperature (Tm).

Repeating this process in the presence of different concentrations of compound (figure 3) allows us to judge whether a compound is thermally stabilizing (i.e., binding to) our protein target or not.

Our instrumentation of choice is the QuantStudio™ 6 Flex Real-Time PCR System which offers high levels of reproducibility.

It facilitates real-time PCR based applications with an upgradable block selection, allowing us to plan and grow as required.

A simplified workflow, intuitive software, and touch-screen interface, enable ease of use for our team.

 

Get in touch to find out more about how we can work with you to apply FTSA for the benefit of your project. 

QuantStudio

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