Protein Thermal Stability Analysis (CETSA)

Cellular Thermal Shift Assay (CETSA) is an experimental technique used to determine the thermal stability of proteins within cells. This technique is based on one core principle, that proteins exhibit different stabilities at different temperatures, which can be determined by detecting whether the protein is still soluble in the cell after heating. CETSA can be used to study the physical properties of proteins, as well as to assess the interaction between drugs and their target proteins. Here are the general steps involved in a CETSA experiment:

 

Analysis Workflow

1. Sample Preparation

First, cell or tissue samples are prepared. These samples can be cultured cell lines, animal tissues, or any other biological material that contains the target protein.

 

2. Treatment and Heating

The samples are usually treated in the presence or absence of small molecule drugs. Afterwards, the samples are heated at a series of incremental temperatures, usually ranging from room temperature to boiling. The purpose of this step is to induce varying degrees of protein denaturation.

 

3. Separation and Detection

After heating, the samples are separated by centrifugation or filtration to separate the soluble proteins from the precipitated ones. Then, protein detection techniques (such as Western blotting, enzyme-linked immunosorbent assay (ELISA), or mass spectrometry) are used to quantify the amount of protein in the soluble phase at different temperature points.

 

4. Data Analysis

By comparing the stability of the protein at different temperatures, a thermal stability curve of the protein within the cell can be obtained. If small molecule drugs were used in the experiment, their effect on the thermal stability of the protein can also be observed.

 

The advantage of the CETSA technique is that it can directly determine the stability of proteins in their natural environment within cells, which is crucial for understanding the real state of proteins in the body and the mechanism of action of drugs. Moreover, CETSA can be used to screen drug targets, aiding in the drug discovery and development process.