A CTG assay, often referred to as a Cellular Thermal Shift Assay (CETSA), is a technique in molecular biology used to investigate how proteins interact with other molecules, such as potential drug compounds, within living cells. This method provides insights into protein stability and changes in their structural integrity when they bind to a ligand. By observing these changes in a cellular environment, researchers gain a more accurate understanding of molecular interactions.
Understanding CTG Assay: What It Is
The CTG assay is based on the principle that the thermal stability of a protein can change upon binding to another molecule. Proteins naturally unfold or denature when exposed to increasing temperatures, leading to their precipitation. When a protein binds to a ligand, its structure often becomes more stable, requiring a higher temperature to induce unfolding. This stabilization, or “thermal shift,” serves as a direct indicator of a molecular interaction occurring within the cellular environment.
This shift in thermal denaturation temperature provides valuable information about whether a compound is binding to its intended protein target inside cells. Unlike traditional methods using purified proteins in a test tube, the CTG assay allows observation of these interactions in a more physiologically relevant context. This cellular context accounts for factors such as membrane permeability, intracellular metabolism, and other cellular components that can influence drug-target engagement. The assay offers a label-free approach to directly assessing target engagement of compounds in cells and tissues.
The Science Behind CTG Assay
Performing a CTG assay involves several steps to observe changes in protein solubility with temperature. Cells are initially treated with a compound of interest, or a control, and then subjected to a gradient of increasing temperatures. As the temperature rises, proteins begin to unfold, become insoluble, and precipitate.
Following the heat treatment, the cells are lysed, and the soluble protein fraction is separated from the insoluble, denatured proteins. The amount of the target protein remaining in the soluble fraction at each temperature point is then measured. Common detection methods include Western blotting or quantitative mass spectrometry. Changes in the amount of soluble protein across the temperature range reveal a protein’s thermal stability profile, allowing researchers to determine if and how a compound affects this stability.
Where CTG Assays Are Used
CTG assays have found broad utility in drug discovery and development. They are routinely employed to identify and validate potential drug targets by confirming that a drug candidate binds to its target protein within living cells. This is particularly useful in early-stage drug development to confirm target engagement, a significant factor in predicting therapeutic effect.
The assay also helps understand the mechanism of action of new compounds, revealing how they interact with targets and influence protein stability. Beyond drug discovery, CTG assays contribute to studying protein-protein interactions and exploring disease mechanisms where protein stability is altered. For instance, it can be used to investigate drug binding in specific cellular environments like platelets, providing insights into how drugs interact with their targets.
Making Sense of CTG Assay Results
Interpreting CTG assay results involves analyzing the protein’s “melting curve,” which plots soluble protein against increasing temperatures. A typical melting curve shows a decrease in soluble protein as temperature rises, indicating denaturation. When a ligand binds to a protein, it often stabilizes the protein’s structure, causing the melting curve to shift towards higher temperatures.
This shift signifies that a higher temperature is required to denature 50% of the protein, known as the melting temperature (Tm). An increase in Tm indicates the compound has bound and stabilized the protein, confirming an interaction. Conversely, a decrease in Tm could suggest that the compound destabilizes the protein. Researchers look for these thermal shifts to confirm binding and understand interaction strength in a cellular context.