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FAQs on CETSA Assays and The Use of Alpha for Detection

Disclaimer: For research use only. CETSA® is a registered trademark of Pelago Bioscience AB. Please note that a valid CETSA® subscription is required for kit use.

The CETSA® method itself

Q : What is CETSA®?

A : The Cellular Thermal Shift Assay is a method that allows the quantification of a compound’s Target Engagement (TE) within living cells or in disrupted cells. The CETSA® assay principle is based on the change in thermal denaturation profile of the target protein that occurs following the binding of a compound. The CETSA® assay is performed by incubating the cells with the test compound, followed by heating of the compound-treated cells, and then by measuring the remaining soluble target protein.

Q : What is Thermal Shift

A: Upon heating a protein will encounter a temperature at which it denatures (sometimes referred to as the melting point). This melting temperature is a physical property and a constant for any given set of conditions (pH, pressure, salts). Compounds that interact with a protein will change the melting temperature (thermal shift). For a given binding site within a protein the size of the thermal shift can be measured at different compound concentrations (dose-response), and the EC50 values derived from such curves can be used to establish a rank order of potency of a series of compounds, which directly correlates to the rank order of affinity of the compounds . There are several variations on the in vitro Thermal Shift assay, but the key technique was first described by Semisotnov et al. (1991). In this method, the melting and unfolding protein exposes hydrophobic surfaces that enable SYPRO orange to bind. This dyes’s fluorescence is quenched in water, so the binding to these hydrophobic surfaces reverses this and causes fluorescence to peak when the protein is fully unfolded. This type of Thermal Shift assay can only be applied to highly purified proteins and for low abundance species this may mean that an over expression system is required to generate sufficient amounts.

Thermofluor – temperature sensitive dye system (using Sypro Orange)

Differential scanning fluorimetry (DSF) – alternative dye based methods

Quantitive PCR systems (eg Roche Light cycler) : these are used to deliver the heating event and can measure fluorescence changes

Nanotemper – is a company manufacturing dedicated instrument that heats the sample and measures the fluorescence. It offers better performance than the adapted PCR systems.

Q : Why is Measuring Target Engagement Important?

A : In the absence of confirmation that a compound indeed interacts with the desired target, drug developers can lose precious time and resources moving in the wrong direction. For that reason, confirmation of a compound’s Target Engagement has long been regarded as essential in drug discovery. Such assays enable direct comparisons of a compound’s affinity for its target to be made, as such it is an essential measure to select which compounds to advance at all stages of lead generation and optimization. Because Target Engagement can be correlated to affinity it enables researchers to select compounds that have the highest affinity values. As such it is an extremely useful measure to ensure the correct compound prioritization at each stage of the drug discovery process.

Q: How does CETSA® differ from other Thermal Shift assay technologies?

A: Beside CETSA ®, there are numerous methods for assessing Target Engagement (e.g. Surface Plasmon Resonance, Fluorescence Polarization and Thermal Shift). However all these methods rely on purified protein being available and can only be applied in vitro, meaning the derived affinity values may not be predictive of actual affinity or binding potential in living cells. Being able to run Thermal Shift assays in a relevant cellular context, where the protein is in its native environment, in the presence of its natural partner proteins, with physiological concentrations of its cofactors and substrates, yields much more relevant data, that will better translate into animal models and clinical trials.

While the Cellular Thermal Shift Assay is a method based on the same biophysical principle as standard Thermal shift assays (i.e. that specific proteins will denature at a set temperature) it does not directly measure the specific temperature of unfolding, but relies instead on the fact that the presence of a compound on the protein will affect the amount of soluble protein present after heating to a set temperature. As such, CETSA® is in essence a total protein assay conducted after a specific heating event. Compounds with differing target engagement potencies will change the relative amounts of protein surviving the heating event. Because the assay measures this residual protein, rather than actual melting event, it can be applied to more complex in vivo systems such as cells and lysates (and in some CETSA® formats even solid tissue). In addition, CETSA® can identify compounds that destabilize a protein (i.e. reduce its melting temperature), this is less straight forward with the other thermal shift methods.

Q: How does Alpha CETSA® differ from other Cellular Thermal Shift assay technologies or other Cellular Target Engagement Assays?

A: As mentioned above, CETSA® is basically a total protein assay conducted after heat shock. Alpha CETSA® uses a dual antibody proximity based detection system. Other methods avoid the use of an antibody based system by modifying the target protein:

  • Promega nanoluciferase thermal shift assay (NaLTSA) : Nanoluc Luciferase Fused to the target protein (recombinantly expressed protein); When the protein target with the Nanoluc enzyme tag aggregates, the luciferase signal will decrease.
  • Promega NanoBRET Target Engagement Assay : Luciferase Fusion Tag combined with labelled tracer compounds which, when in close proximity to the luciferase will lead to Bioluminescence Resonance Energy Transfer (BRET). Compound binding in the same pocket will displace the tracer and BRET signal will decrease. This is Not a heat shock method.
  • DiscoverX InCELL Pulse : Based on Enzyme Fragment Complementation (EFC) technology : the target protein is fused with a small enzyme donor fragment of β-galactosidase (β-gal). An added reporter protein will bind to this fragment to reconstitute an active enzyme, which will hydrolyze a substrate and generate a chemiluminescent signal allowing protein abundance quantification. After heat denaturation, the protein will aggregate and limit access of the larger luciferase component to its partner on the protein target.

The key difference between these “recombinant CETSA® ” or “recombinant target engagement” systems and Alpha CETSA®, is that they cannot be applied to native and unmodified cells. This has a number of negative consequences:

  1. While the cost of developing a novel antibody pair and the subsequent per well costs may be higher than simply transfecting a single cell line, it is likely that any discovery program will want to be tested with a variety of model cell lines, each transfection comes with its own costs and cloning out the subsequent cell lines will take additional weeks.
  2. Generating a tagged protein will always have physiological consequences on the cell and the tagged protein may be (i) overexpressed (wrong stoichiometry vs. partners), (ii) not located in the correct cellular compartment or (iii) not associated with key partner proteins and (iv) may have a different melting behaviour than the native protein. Meaning that the apparent effect of a compound may not reflect its real behavior in patient’s tissue. These problems may be magnified in any temporary transfection system.
  3. Luciferase inhibitors may result in false positive hits.
  4. In later stages of lead optimization when more complex and physiologically more relevant cellular models (primary, native cell lines and tissue) are required, tagged protein approaches are simply not applicable.

Q: What information does a CETSA® assay deliver?

A: CETSA® provides a unique measure of a compounds ‘on target presence’ and can be thought of as target occupancy. This occupancy will be affected by both the compounds ability to engage the target and the ability of the compound to be present at the right location (i.e. does it have the right solubility, permeability, metabolic stability and availability at the right cellular compartment). Non-cellular Target Engagement assays offer measures of affinity that correlate only with the protein’s behavior in highly artificial environments often using purified recombinantly expressed target proteins.

Q: Can CETSA® be used for SAR analysis studies?

A: There are no published examples yet where CETSA® has been used for compound optimization. However, the potential and applicability of CETSA® to be used for SAR analysis and hit confirmation were clearly demonstrated by Shaw et al. by the comparison of data from the CETSA® assay with commonly used biochemical and cell-based assay data (Shaw et al. 2018 SLAS Discovery 1-12 DOI: 10.1177/2472555218813332). The publication demonstrates the added value of CETSA® for screening, hit confirmation, and SAR generation for two protein targets: B-Raf and PARP1.

Target/Sample Types

Q: How many targets have been validated in CETSA® so far?

A: In CETSA® HT (most being Alpha CETSA® , some using HTRF) more than 30 targets have been successfully validated, including targets with nuclear, mitochondrial, cytoplasmic and plasma membrane localization.


More than 100 targets have been validated so far in CETSA® Classic (Western Blot based detection).

CETSA® M/S is generating information for 6000 to 7000 targets at once.

Q: Can all target proteins be tested in CETSA®?

R: Some targets are “blind” in CETSA® , i.e. compound binding will not change their thermal stability. This may happen when the protein has multiple domains, and the compound is binding to one domain only, and if stabilization of this single domain is not globally impacting the thermal stability of the whole protein. There are a few proteins that do not melt in typical temperature range applied in CETSA® experiments.

Pelago has access to a large database with thermal stability data from projects with mass spectrometric readout (CETSA® MS), and this information is taken into account when Pelago is assessing the “CETSA® bility” of a protein target before developing an Alpha CETSA® assay. Please contact Pelago to request such information.

Q : Are GPCRs amenable to CETSA® ?

A: A few examples of GPCRs have been tested in CETSA® . In a recent publication from Astra Zeneca (Kawatkar et al Chem Biology 2019) CETSA® Classics assays were established for a set of membrane bound proteins including a GPCR protein target. One potential challenge with CETSA® on GPCRs may be limited availability of antibodies for detection. In addition, the integral membrane localization of the GPRCs can lead to unpredictable melting behavior.

Q : What type of samples can be tested in CETSA®?

A : There are examples of CETSA® assays using many different types of matrices including immortalized cell lines, primary cells, iPS cells, patient-derived PBMCs, spheroids, nuclear fractions from cultured cells, ex-vivo treated tissue, tissues from in vivo animal studies, bacteria, yeast, zebrafish and insects.

Q : Can the samples be frozen after the heating step?

A : This is possible, however this requires a case-by-case validation as the freezing process can denature some proteins.

Q: My cells need culture plate coating with some proteins (e.g. collagen or Matrigel); is special care needed to avoid sample preparation?

A: We have not encountered any issues with cells cultured on coated plates.

CETSA® Assay Conditions

Q: What are the typical Alpha CETSA® assay optimization points?

A: If starting from scratch, the immunoassay needs to be developed and optimized first. It is important and worthwhile investing on developing a very sensitive Alpha assay, as it will allow to reduce the cell number per well needed in the CETSA® assay. Because the CETSA® assay is destroying part of the protein that needs to be detected, typically more cells/well are needed in a CETSA® assay compared to other cell-based assays. Please refer to the PerkinElmer guides for alpha assay development for how to proceed and for useful hints, and to the CETSA® toolbox manuals if using the toolbox (anti mouse Ig X anti rabbit Ig beads) approach.

Then the CETSA® assay optimization points include:

  • Cell density titration
  • Selection of cell lysis buffer
  • Incubation time of cells with the test compound
  • Temperature for incubation of cells with the test compound
  • Establishing melt and shift curves
  • Selection of temperature for isothermal dose-response experiments
  • Workflow and automation settings

Q: Why are different CETSA® cell lysis buffers provided and what is the impact of using different cell lysis buffers?

A: The lysis buffer is important regarding several aspects of the assay. Its role is multiple: lysing the cells and potentially free the target protein from partner proteins that may interfere with antibody recognition, in order to make the target available for detection by the antibodies; stabilizing the target protein to make the assay stable; avoiding or minimizing potential epiturbance effect; providing the right physico-chemical conditions (pH, ionic strength and nature of salts, detergent type and concentration, … ) for the immunoassay, … As different immunoassays may have different optimal conditions, and as different protein targets, and different cell types may have different requirements for optimal assay performance, we are making 5 different cell lysis buffers available. These provide a variety of cell lysis conditions. However this does not imply that additional components may be added in specific cases, like additional detergent types, to improve further assay performance.

Q: Are there protease inhibitors in the CETSA® cell lysis buffers?

A: CETSA® Cell Lysis buffers 1, 4 and 5 contains some divalent cations chelators, which are expected to inhibit proteases requiring such divalent cations for their activity (Matrix Metalloproteinases for example). Besides this, no other protease inhibitors are included in the CETSA® cell lysis buffers as these are generally not needed for the performance of Alpha CETSA® assays. However for specific cell types or tissue samples, you may want to add typical proteinase inhibitors, such as leupeptin.

Q: What are the typical melting temperatures?

A: The melting temperature (Tm) is defined as the temperature at which half of the target protein population has been denatured (i.e. in Alpha CETSA® at which half of the Alpha signal has been lost). Depending on the target, melting temperatures is most often between 40°C and 60°C, with an average melting temperature of 52°C. Some proteins, like membrane associated proteins, are usually more stable and can have a higher melting temperature. CETSA® assay data for proteins having a melting temperature above 65°C may be affected from the fact that the membrane permeability and cell integrity is perturbed when cells are heated, thereby impacting the physiological significance of the assay.

In our experience, the melting temperature is target-specific and depend of the assay matrix and of the lysis buffer used.

Q: Is the Tm expected to be the same when working with intact cells and disrupted cells?

A: Not necessarily, as when disrupting cells, protein-protein interactions that stabilize proteins can be lost, which may lead to a changed melting temperature as compared to intact cells. For an example please refer to the Alpha CETSA® MEK1 assay data.

Q: What heating temperature should I select for compound testing?

A: For stabilizing compounds it is recommended to select the lowest temperature with the largest assay window, as it is expected to give the most sensitive assay (lower EC50 values). For destabilizing compounds it is recommended to select the highest temperature with the largest assay window. Temperatures above 65°C can have an impact on the cellular permeability and reduce the significance of the data.

Q: Should I expect the same melting temperature in CETSA® Classic (Western Blot) and Alpha CETSA®?

A: Not necessarily: the apparent melting temperature can vary between both methods, because the detection methods are different.

Q: Is it important to have a strict control of sample heating times?

A: Yes this is extremely important to control as longer heating times may result in a right shift of the dose-response curve (see below the comments about residence time). The standard heating time is 3 minutes.

Compounds with a short residence time (i.e. high dissociation rate constant koff; residence time T being equal to 1/koff) will dissociate faster from the target when it is heated, compared to compounds having a long residence time. For that reason, the EC50, or ITDRF50 value is expected to increase with increasing heating times. For more explanations on the CETSA® theory see Seashore-Ludlow B, Axelsson H, Almqvist H, Dahlgren B, Jonsson M, Lundbäck T. (2018) Quantitative Interpretation of Intracellular Drug Binding and Kinetics Using the Cellular Thermal Shift Assay. Biochemistry 57:6715-6725. In theory, detection of different compounds residence times at the target, by varying heating time could be possible but there are no published reports yet available about this having been done.

Q: The manual instructs to either cool on ice, or for 3 min at 25°C after the heat shock. Is this cooling step important and is it important to have a strict control of the cooling time?

A: Cooling the sample rapidly ensures that the heat shock phase is well controlled. Simply allowing the temperatures to cool unaided would certainly produce different cooling rates in different wells and affect the amount of heat shock delivered to the system. The cooling phase is not as critical as the heating phase but should be conducted quickly and consistently.

Q: May I use PerkinElmer “Biomarker” and “SureFire” (non-CETSA® ) kits to perform a CETSA® assay?

A: In theory any total protein assay may be suitable for use in a CETSA® protocol, though each system needs to be optimised and validated. However, the CETSA® method is patented and permission will be needed from Pelago Bioscience. Furthermore, only the use of the designated target kits are supported by PerkinElmer. Use of the tool box kit is supported by Pelago Bioscience and is only available to license holders.

Q: Is it possible to read the Alpha signal directly from the PCR plate used to heat the samples?

R: This would provide advantages in terms of number of pipetting steps, sample consumption and ease of automation but the possibility of using PCR plates for the full process – from sample treatment to detection – is not demonstrated yet. PCR plates are often very flexible, leading to non-uniform signal over the plate, or do not have the right dimensions to allow them to be handled by plate readers. Well-to-well cross-talk can also be an issue in such PCR plates.

Q : Can chemical denaturation (e.g. using urea or guanidine) be used instead of heat?

A: The advantage of using temperature to deliver the heat shock is that it is done in a highly controlled way that is likely to be constant between samples, and limited over a well-controlled time frame. A chemical denaturation process might work in a purified protein extract; but in the complex cellular environment, variation in cell volume, buffering capability, lipid content etc are all likely to mean that different cells lines display different denaturation characteristics for any given protein.

As denaturation (heating time) is critical, this may be quite difficult to control if using chemical denaturation. In addition heat can be applied without disrupting cells, allowing to test in real cellular context. This would not be the case of chemical denaturation, as cells would have to be disrupted to allow target access to the denaturating agent, therefore losing intracellular concentrations, protein associations etc.. Therefore we do not recommend replacing the heat shock by chemical denaturation.

Alpha CETSA® Immunoassay

Q: When developing a CETSA® assay, do I need monoclonal antibodies or can polyclonal antibodies be used as well?

A: Both monoclonal or polyclonal antibodies can be used, depending of course on the quality of the antibodies. Monoclonal antibodies present an advantage in terms of stable reagent supply, as for any other detection method using antibodies.

Q: When using polyclonal antibodies, will the next lot work the same way in a CETSA® assay?

A: In our experience, we never faced a situation where the next lot of a polyclonal antibody behaved differently in the CETSA® assay compared to former lots. However, the CETSA® assay needs to be re-validated with the next lot of the polyclonal antibody.

Q: Are there other recommendation for antibody selection?

A: If available, antibodies should be selected so they cover different epitopes of the target protein, in order to maximize the chances to find a suitable antibody pair that will work in the Alpha CETSA® assay.

Antibody pairs yielding steeper curves (higher Hill slope) and less background are preferred as they usually provide a more specific signal. Low Hill slope may be the sign of the ability of an antibody pair to recognize the re-folded protein.

Q: Are the Alpha CETSA® antibodies against the endogenous target protein only or the endogenous target protein plus bound small molecule?

A: Based on the kits developed so far, the antibodies are against the protein target only.

Q: Can I expect detection antibody affinities to be different between bound and unbound small molecule to target protein?

A: Small molecules influencing protein folding at epitope sites are indeed at risk of altering antibody binding. This phenomenon is called “epiturbance” and can be a limitation of antibody based-CETSA® . Epiturbance is in general not observed in the CETSA® Classic assay format (Western Blotting) as in this case the protein is fully denatured before the antibody detection step.

Q: Can only IgG be used with the CETSA® toolbox kits?

A: Indeed, the antibodies on the anti-rabbit and anti-mouse beads are specific to IgG, and would not recognize other antibody classes (i.e. IgA, IgM, etc should not be selected).

Q: Is there an advantage in using Alpha CETSA® compared to CETSA® Classic (Western Blotting)?

A: Beside greater sensitivity of the Alpha CETSA® assay and throughput and automation considerations, due to the possible difficulty in analyzing membrane proteins in the CETSA® Classic method, Alpha CETSA® may perform better for such targets, as it does not need any separation step.

CETSA® Data Analysis and Interpretation

Q: What false positive hits can be obtained in CETSA®?

A: The thermal stability of some proteins can be affected following compound treatment, even though they are not directly bound by the test compound. These indirect effect can be the result of for example protein phosphorylation, protein recruitment by a partner protein, or from general change of the Redox state of the cell. Running the CETSA® assay in parallel on intact cells and on disrupted cells can discriminate between direct and indirect effects, as such indirect effects are not expected when the cells are disrupted and the metabolic processes are stopped.

Compound assay intereference can be encountered in Alpha CETSA® since the compounds are present in the detection step at a relatively high concentration. This can give misleading results and it is important to perform a counter screen to identify these compounds.

Q: How can target destabilization be interpreted?

A: Destabilization can result from the disruption by the compound binding of a protein complex that was stabilizing the target protein. In our experience, membrane proteins usually have a higher melting temperature and are rather destabilized than stabilized by compound binding. For kinases, it could as well result from the displacement of ATP. In such case, comparing the result of CETSA® assays when performed on intact cells (physiological ATP concentrations) and disrupted cells (loss of ATP) can be useful. For some targets (see the ErbB2 Alpha CETSA® kit manual) we have observed that irreversible inhibitors were destabilizing the target while a reversible inhibitor was stabilizing the target.

Q: Is there a way to avoid epiturbance effect?

A: Adding a small amount of detergent, like 0.01% SDS, may prevent the epiturbance phenomenon. An explanation could be that SDS is providing access to the antibody even in the presence of the compound. Some of the CETSA® cell lysis buffer contain a small amount of SDS and therefore may avoid epiturbance over other lysis buffers. We cannot exclude that more SDS may need to be added in some cases.

It is to be noted that such an epiturbance effect is not limited to CETSA® assays, but can also be encountered in any immunoassay.

Q: Counter-screen / How to check if my CETSA® hit is a false positive?

A: In CETSA® , there is a simple method allowing to determine if the compound is acting on the target protein (de)stabilization itself, or is interfering with the detection method: a comparison can be made between (1) addition of the compounds to the cells, then incubating and doing the heat treatment and (2) heating the cells first and adding the compound only after. If the compound activity is found only in test 1, then it is truly active at the target. If compound activity is found in 1 and 2, then it shows that it interferes somehow with the detection technology (refer to the PerkinElmer Protein-Protein interaction guide for a list of potential alpha interferences).

Q: What false negative hits can be obtained in CETSA®?

A: If a compound is not reaching the target in a cellular context, it may appear as positive in biochemical assays, and still be negative in CETSA® . This is not a false negative, but just reflecting the fact that the compound is not able to access the target under real cellular conditions, which is part of the powerfulness of the method.

Q : How do CETSA® values compare with functional assays / is there an meaning of the thermoshift amplitude?

A: When analyzing CETSA® data, one must keep in mind that the principle of the assay is quite different from typical functional assays, such as looking at protein phosphorylation, ion concentrations, cAMP and other signal transduction markers, or phenotypic analysis in high content screening, and therefore the data must be interpreted accordingly.

The amplitude of the thermoshift is NOT a measure of compound affinity.

The EC50, or ITDRF50 values, are specific to certain assay conditions (temperature, time of heating, …). This is not different from functional assays, where EC50 values are for example specific to stimulation times.

Q : Is it possible to discriminate antagonists vs agonists in CETSA® assays?

A: Normally this is not an information that is extracted from CETSA® assays, but the publication from Shaw et al. (2018) Scientific REPORTS | (2018) 8:163 | DOI:10.1038/s41598-017-18650-x. reports that only agonists were able to stabilize the androgen receptor in the CETSA® assay developed, while antagonist had no direct effect in the CETSA® assay, but could be detected as competitors of the effect of agonists in the CETSA® assay. Therefore in this particular case the CETSA® assay was able to discriminate between androgen receptor agonists and antagonists. This does not imply that this is possible for any target, as there are numerous examples where the CETSA® assay directly detects both agonists and antagonists.

Q: Can CETSA® be used to detect toxicity of a compound?

A: Compounds exerting some toxic effect on a cell are expected to lead to change in the level of some proteins (via degradation and/or transcriptional effects) and to changes in thermostability of some proteins (via post-translational modifications or general change of the Redox state of the cells). Pelago is exploring the possibility to generate “CETSA® fingerprints” that could be linked to different types of toxicity, from CETSA® MS data. This could also generate knowledge of targets that drugs should not hit to avoid such toxicity. If a specific target would appear to be linked to toxicity, then use of Alpha CETSA® may become a method of choice for the pharma industry.

Q: Can a housekeeping protein be used for normalization of CETSA® assays?

A: Normalization is typically not needed in CETSA® assays, as the important output of the assay is the EC50 value. However, in some cases, for example when working with tissue samples, the quantity of material engaged per datapoint may vary quite significantly and normalization may then be desired. In CETSA® Classic (Western blotting), SOD1 is commonly used as its melting point of 80°C makes it a good invariable reference for any target, and as its molecular weight of 18 kDa makes it easy to detect on Western blots. GAPDH would not be recommended due to its lower melting temperature (55 °C), making it not a possible control for targets with a higher melting temperature.

If wanting to normalize Alpha CETSA® assay, cofilin could be tried (there is an AlphaLISA SureFire ultra kit for total cofilin, with preliminary CETSA® data but no fully validated kit yet)