Question

Thermal shift Assay (CESTA)

1. Explain the concept behind CESTA. What is positive results, what is negative results? What does it mean when there is a “shift”?

Answer 1

Thermal shift assay is CETSA and not CESTA. Thermal shift assays detect protein-drug interactions in therapeutic samples. It can be carried out on cells samples or microtiter format. CETSA relies on protein stabilization after binding of ligand.

When an unbound proteins are exposed to gradient of temperatures, they will unfold or melt at a particular temperature. The parent melting temperature is the mid-point of the transitions where 50% of the proteins have denatured or unfolded. However, when a protein is bound to a ligand, it will melt at much higher temperature than normal due to more stabilization. This is known as Tm shift. The term Tm shift is normally associated with equilibrium processes. However, in CETSA, remaining levels of stabilized proteins are quantified after the thermally unfolded proteins are irreversibly aggregated. This change is a c=non-equilibrium change. Hence, the shift is known as thermal aggregation temperature (T_agg) shifts.

The protocol is as follows (general):

1) The drug treated sample (lysate or whole cell or tissue) is transiently heated till cells/proteins denature. All proteins that are not stabilized by ligand are precipitated.

2) The sample is cooled and the cells are lysed.

3) precipitated protein can be removed if required. The remaining non-precipitated soluble proteins are detected in the supernatant.

Positive Results: The Delta Tm will increase for the protein + drug vs the protein sample.

Negative results: The delta Tm will remain the same for protein + drug or protein sample.

Shift is the change in Tm of the proteins when bound to the ligand (drug). If there is a shift, it means there is drug protein interaction.