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Short answer
CETSA (cellular thermal shift assay) measures whether a compound binds a protein inside intact cells by checking how the protein’s thermal stability changes. A protein bound to a ligand usually unfolds and aggregates at a different temperature than the unbound protein, so the amount of protein left soluble after heating reveals binding. The main formats are melt-curve (temperature range) and isothermal dose-response experiments.
Definition and scope
CETSA is a target-engagement method: it asks whether a molecule physically reaches and binds its protein target in a cellular setting, rather than only in a purified-protein tube. In the words of a Scientific Reports paper on a split-NanoLuc version of the assay, CETSA “enables such an assessment by quantifying the changes in the thermal stability of proteins upon ligand binding in intact cells.” Heated proteins unfold and aggregate at a characteristic temperature, reported as Tagg (or Tm), and ligand binding can shift that value.
The method’s developers describe it on cetsa.org as the first broadly applicable method to assess direct drug binding in cells and tissues, and say they published the first paper in 2013. The assay is a measurement of binding-related stability, not of what a compound does downstream: it does not by itself show a functional effect.
How CETSA is used in practice
Every CETSA format follows the same logic: expose cells (or lysates) to a compound, heat them, then measure how much of the target protein stays soluble. In the traditional format, heated cells are processed so that unfolded and aggregated proteins are removed by centrifugation, and the remaining soluble protein is measured by a protein detection method such as western blot. Mass spectrometry has been used to detect a substantial portion of the entire melting proteome.
Two experiment designs answer different questions:
- Temperature-range (melt curve) experiment: cells are heated across a range of temperatures at fixed compound concentration. The question is whether the Tagg of the target moves when the compound is present.
- Isothermal dose-response experiment: cells are heated at one temperature across a range of compound concentrations. The question is how much compound is needed to stabilise the target; the result is expressed as an AC50. The same paper notes that AC50 values are right-shifted with increasing heat time, so the heating duration is part of the result and has to be reported with it.
Readout formats compared
The detection step is where the formats differ most.
| Readout | Reported detail | What it changes |
|---|---|---|
| Western blot | Traditional detection of the soluble protein left after aggregated protein is removed by centrifugation | Antibody-based, one protein at a time; centrifugation step present |
| Mass spectrometry | Has been used to detect a substantial portion of the entire melting proteome | Reads many proteins at once instead of one chosen target |
| Split NanoLuc (HT-CETSA) | Cells are lysed with 1% NP40; centrifugation was not required for that assay | Plate-based luminescence readout; target carries a tag |
| RT-CETSA (NIH) | Monitors aggregation in cells in real time across a range of compound concentrations; works in intact cells and lysates | NIH technology-transfer listing says traditional CETSA is run at a single temperature (dose response) or a single concentration across temperatures, while RT-CETSA captures both in one experiment |
The RT-CETSA advantages, including more sensitive and reproducible hit identification, are the inventors’ claims as stated in the NIH Office of Technology Transfer listing.
What CETSA can miss: a checklist
The split-NanoLuc paper in Scientific Reports notes these limits, which apply when reading any CETSA result:
- Not all proteins are expected to demonstrate a thermal shift when a compound binds, so a compound that binds without a shift would be missed (a negative is not proof of no binding).
- A fusion tag, such as the one used in a luciferase-fragment format, might alter the protein’s thermal stability.
- Experiments should be interpreted with caution when heating above about 55 C because of effects on membrane integrity in intact cells.
- Heating time moves the apparent AC50, so isothermal dose-response numbers are only comparable at matched heat times.
Where it matters for labs and supply decisions
The same stages appear in every format: a heating step, a lysis step in cell-based formats, a separation step in the traditional format, and a detection format (western blot, mass spectrometry, or a luminescence-based readout). A lab planning to run CETSA therefore needs to decide the detection format first, because it determines whether a centrifugation step is required and whether the target must be tagged. The assay is also one of several ways to ask about binding; a lab that wants binding kinetics on purified material can look at bio-layer interferometry instead.
Related bench methods on CASRAI: western blot loading-control normalisation for the traditional readout, mass spectrometry proteomics sample preparation for the proteome-wide readout, luciferase reporter assays for luminescence detection, and qPCR melt curve analysis for another thermal-denaturation readout.
Common misunderstandings
- “A thermal shift proves the compound works.” It indicates binding-related stabilisation of the protein, not a functional effect.
- “No shift means no binding.” Not every protein is expected to show a thermal shift when a compound binds.
- “CETSA is only a western blot assay.” Western blot is the traditional readout; mass spectrometry, split-luciferase and real-time formats are described in the literature.
- “A melt curve and a dose-response curve are interchangeable.” The first reports a temperature shift at fixed compound concentration; the second reports potency at fixed temperature.
FAQ
What does CETSA measure?
CETSA quantifies changes in the thermal stability of proteins upon ligand binding in intact cells, which indicates whether a compound binds its target there.
What is the difference between a melt-curve and an isothermal dose-response CETSA experiment?
A melt-curve (temperature-range) experiment heats cells across temperatures to see whether the target’s Tagg shifts. An isothermal dose-response experiment heats at one temperature across compound concentrations and reports an AC50.
Does a lack of thermal shift mean a compound does not bind?
No. Not all proteins are expected to demonstrate a thermal shift with compound binding, so a binder without a shift would be missed.
Does CETSA always need a western blot?
No. Western blot is the traditional readout; mass spectrometry, a split-NanoLuc luminescence format and the real-time RT-CETSA format are also described.
Sources
- Split NanoLuc high-throughput CETSA paper, Scientific Reports, PubMed Central article PMC6013488: pmc.ncbi.nlm.nih.gov/articles/PMC6013488 (checked 2026-10-29).
- CETSA developers’ site, About page: cetsa.org/about (checked 2026-10-29).
- NIH Office of Technology Transfer, RT-CETSA listing: techtransfer.nih.gov/tech/tab-3525 (checked 2026-10-29).
