Thermal Shift Assay Services

Thermal shift assays measure the thermal stability of a target protein and the increase in protein melting temperature upon the binding of a stabilizing agent. It is useful for identifying ligands, buffer conditions, and cofactors affecting protein stability. 

Reaction Biology performs Differential Scanning Fluorimetry using a qPCR machine for high-throughput data acquisition in the 384 well format. We offer thermal shift assays for the reader domain proteins, including bromodomains, chromodomains, and other methyl-readers. In addition, we offer custom-tailored assay development for other targets.

  • Useful to identify ligands, buffer conditions, and cofactors
  • Fast setup and minimal assay establishment requirements
  • Deliverable: melting temperature shift (∆Tm) as a readout for compound binding affinity

TSA Principle

TSA measures the melting temperature of a protein (Tm), which is the temperature at which there is 50% denaturation. Protein denaturation is monitored via an  increase in fluorescence of SYPRO Orange dye which binds to hydrophobic residues that get exposed as the target protein unfolds.

thermal shift assay principle based on protein denaturation and binding of fluorescent dye to the unfolded protein for determination of melting temperature

Assay Details

Example: cGAS

thermal shift assay example with differential scanning fluorimetry for determination of affinity binding of a compound to a target

Example of Differential Scanning Fluorimetry of cGAS in the presence or absence of 10 µM PF-06928215.

The thermal denaturation of cGAS is detected via SYPRO Orange dye. The addition of the inhibitor PF-06928215 stabilizes the protein and increases the melting temperature from 45 to 53 degrees Celsius.

Example: BRD2

example of a thermal shift assay graph with differential scanning fluorimetry to determine the binding affinity of a compound to a target performed with BRD4 bromodomain contstruct

Differential Scanning Fluorimetry of BRD2-Tndm in the presence or absence of (+)- JQ1. The thermal denaturation of BRDT-Tndm is detected via SYPRO Orange dye. Addition of the BET Bromodomain inhibitor/ligand (+)- JQ1 stabilizes the protein folding and shifts the Tm from 41 to 44 degrees Celsius.

Screening Details

Instruments: BCFX384 (BioRad) real-time PCR detection system

Turnaround time: The study is typically completed within 2 weeks after receipt of materials.

Projects are performed on a first-come-first-serve basis.

Sample requirements: Protein purity of 85% or more is required. The protein amount is project dependent. Typical usage is between 1-3 ug per reaction.
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Screening location: The assay is performed in Malvern, PA, USA.