Application: The Alpha CETSA® kit presented here is intended for the quantitative determination of a soluble target protein of choice in Cellular Thermal Shift Assay (CETSA®) using a homogeneous AlphaLISA assay (no wash steps). The assay kit contains AlphaLISA acceptor beads conjugated to an anti-rabbit IgG antibody (Fc specific) for the capture of a rabbit IgG antibody, and Alpha Donor beads conjugated to an anti-mouse IgG antibody (Fc specific) for the capture of a mouse IgG antibody. The rabbit and mouse antibodies are not provided in the kit and have to be selected by the user, according to the antibody selection guidelines below.
Storage: Store kit in the dark at +4˚C. Stability: This kit is stable for at least 6 months from the shipping date when stored in its original packaging and
the recommended storage conditions.
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CETSA® Assay Principle
The Cellular Thermal Shift Assay (CETSA®) assesses the thermal stability of proteins in living cells and cell lysate based on denaturation and aggregation upon heating. The relative amount of remaining soluble protein after heating can be measured, and a thermal melting curve of a known target protein can be generated. Compound binding often affects the thermal stability of proteins, and the shift in the melting curve is indicative of cellular target engagement. Target engagement by compound binding can result in thermal stabilization of the protein target, leading to a right-shifted thermal denaturation profile (as exemplified in Fig 1), but can also result in protein destabilization, then leading to a left-shifted thermal denaturation profile.
The CETSA® assay is run by incubating intact cells or disrupted cells with the test compound and the assay will reflect the ability of the compound to interact with the target protein in a cellular context. In the case of intact cells, the CETSA® assay data is affected by cellular metabolism and permeability. The CETSA® assay takes into account the complexity of the cellular context, and as such provides very valuable and physiologically relevant target engagement information.
Typically, a melting curve is first generated, where the sample, in the presence and in the absence of a reference compound, is heat challenged at 12 different temperatures. From the melting curve a single temperature is selected, where about 80-90% of the target signal is lost, and where there is an obvious shift of the reference compound. This single temperature is then used to perform single concentration or concentration-response curve compound screening.
The concentration-response experiment determines the potency as the concentration yielding 50% of the maximal stabilization or destabilization effect (EC50) at a single selected temperature. This EC50 value is in the literature also sometimes referred as “isothermal dose−response fingerprint” (ITDRFCETSA), to signify its known dependence on assay conditions. The CETSA® assay EC50 can be used for ranking of compounds and reflects the direct compound binding to the native protein target in a cellular context. This data can be used for SAR analysis and correlation with other assay data.
Figure 1 : Principle of the CETSA® melting curve, and thermal stabilization by compound binding.
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Description of the Alpha CETSA® ToolBox Assay
The Alpha CETSA® Toolbox assay kits allow the rapid, sensitive and quantitative detection of a target protein of choice remaining soluble after heat treatment of compound-treated cells. In an Alpha CETSA® Toolbox assay, a mouse Antibody binds to the anti-mouse IgG Donor beads, while a rabbit Antibody binds to the anti-rabbit IgG Acceptor beads. In the presence of the analyte, the beads come into close proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfers in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm (Figure 2). This Alpha signal generated upon illumination of Donor Beads can be read with an Alpha-enabled plate reader, such as the EnVision® Multilabel Plate Reader, the EnSight™ or the Victor® Nivo™ Multimode Plate Readers. The amount of light emission is directly proportional to the amount of soluble protein present in the sample. A.
B.
Figure 2. Principle of detection of the soluble protein by the Alpha CETSA® assay. Only the soluble protein will lead to the generation of an Alpha signal (A), while the thermally denaturated insoluble protein does not generate the proximity needed for an Alpha signal (B).
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License
Please note that CETSA® is a registered trademark of Pelago Bioscience AB who hold the exclusive rights to the CETSA® patent family. If you do not have yet a valid license, please contact Pelago Biosciences at [email protected] to discuss your needs.
The CETSA® method is patented in the following territories
United Kingdom: Reg.no. 2490404 US: Reg.no. 8969014, 9523693 and 9528996 Singapore: Reg.no. 194137 China: Reg.no. ZL201280025677.X Korea: 10-1940342 Hong Kong: India:
Finland, France, Great Britain, Ireland, Italy, the Netherlands and Sweden with Reg.no. 2699910
The granted and pending patents are based on patent application PCT/GB2012/050853.
Precautions
• The Alpha Donor beads are light-sensitive. All the other assay reagents can be used under normal light conditions. All Alpha assays using the Donor beads should be performed under subdued laboratory lighting (< 100 lux). Green filters (LEE 090 filters (preferred) or Roscolux filters #389 from Rosco) can be applied to light fixtures. The Donor Beads should NOT be used under red/orange light as can be found in photographic work darkrooms, as red light (680nm) excites the beads.
• All blood components and biological materials should be handled as potentially hazardous.
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Kit Content: Reagents and Materials
Kit components CETSA-TBX2-A500 (500 assay points)
CETSA-TBX2-A10K1 to 5 (10 000 assay points)
CETSA® Cell Lysis Buffer 1 (5X) * 10 mL, 1 small bottle 2 X 100 mL, 2 large bottles (-K1 kit)
CETSA® Cell Lysis Buffer 2 (5X) * 10 mL, 1 small bottle OR 2 X 100 mL, 2 large bottles (-K2 kit)
CETSA® Cell Lysis Buffer 3 (5X) * 10 mL, 1 small bottle OR 2 X 100 mL, 2 large bottles (-K3 kit)
CETSA® Cell Lysis Buffer 4 (5X) * 10 mL, 1 small bottle OR 2 X 100 mL, 2 large bottles (-K4 kit)
CETSA® Cell Lysis Buffer 5 (5X) * 10 mL, 1 small bottle OR 2 X 100 mL, 2 large bottles (-K5 kit)
AlphaLISA® Immunoassay Buffer (10X)** 10 mL, 1 small bottle 2 X 100 mL, 2 large bottles
* The choice of the Cell Lysis Buffer is an important part of the optimization of the CETSA® assays, and therefore 5 different Cell Lysis Buffers are provided in the 500 datapoints kit to experimentally determine which buffer is optimal for a particular cellular context. Once the optimal buffer has been found, 5 different versions of the 10000 datapoint kits are available, each containing a different Cell Lysis Buffer. The final number in the part number of these kits indicates which Cell Lysis Buffer is included in each of these kits. For example the CETSA-TBX2-X10K1 part number indicates that the kit contains CETSA Cell Lysis Buffer #1, and the CETSA-TBX2-X10K3 part number indicates that the kit contains CETSA Cell Lysis Buffer #3. Extra CETSA Cell Lysis buffers can be ordered separately (cat # CETSA-BUF1-100ML, CETSA-BUF2-100ML, CETSA-BUF3-100ML, CETSA-BUF4-100ML, CETSA-BUF5-100ML).
** Extra buffer can be ordered separately (cat # AL000C: 10 mL, cat # AL000F: 100 mL).
Sodium azide should not be added to the stock reagents. High concentrations of sodium azide (> 0.001 % final in the assay) might decrease the Alpha signal.
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The CETSA® Cell Lysis Buffers 1 to 5 all contain a proprietary mixture of pH buffers, detergents, and salts. The different CETSA® Cell Lysis Buffers each use different types and concentrations of detergents, contain various concentrations of glycerol, or no glycerol, contain divalent cation chelators or not, contain different types of pH buffers and range from pH 7.0 to 7.5, and contain different types and concentrations of salts, in order to provide a variety of Cell Lysis Buffers so that an optimal one can be found for each target or cell type. The optimal Cell Lysis Buffer for a particular type of sample and target will need to be tested on a case-by-case basis.
The CETSA® Cell Lysis Buffers do not contain protease inhibitors as they are commonly not needed to perform CETSA® assays; however, when working with sample types particularly rich in proteases (such as pancreatic cells) the addition of protease inhibitors to the CETSA® Cell Lysis Buffers may be considered.
The table below highlights key differences between the Lysis Buffers.
Cell Lysis Buffer # Key Considerations
CETSA® Cell Lysis Buffer 1 Most aggressive detergent formulation, contains divalent cation chelators, low salt concentration, moderate glycerol concentration
CETSA® Cell Lysis Buffer 2 Less aggressive detergent formulation; optimized for lysis of a broad range of cells without releasing nuclear DNA and minimally disrupting protein interactions; physiological salt concentrations
CETSA® Cell Lysis Buffer 3 Medium detergent concentration, strongest pH buffering capacity, no glycerol, low salt, 0.1% casein
CETSA® Cell Lysis Buffer 4 Medium-High detergent concentration, contains divalent cation chelators, close to physiological osmotic strength, no glycerol
CETSA® Cell Lysis Buffer 5 Medium-High detergent concentration, contains divalent cation chelator, close to physiological osmotic strength, highest glycerol concentration
Please note that the CETSA® Cell Lysis Buffer 5 may turn yellow over time, which has no impact on assay performance.
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Recommendations
General recommendations:
• The volume indicated on each tube is guaranteed for single pipetting. Multiple pipetting of the reagents may reduce the theoretical amount left in the tube. To minimize loss when pipetting beads, it is preferable not to pre-wet the tip.
• Centrifuge all tubes (including lyophilized analyte) before use to improve recovery of content (2000g, 10-15 sec). • Re-suspend all reagents by vortexing before use. • Use Milli-Q® grade H2O (18 MΩ•cm) to dilute 10X AlphaLISA ImmunoAssay Buffer. • When diluting the samples, change tips between each sample dilution. When loading reagents in the assay microplate,
change tips between each sample addition and after each set of reagents. • When reagents are added to the microplate, make sure the liquids are at the bottom of the well. • Small volumes may be prone to evaporation. It is recommended to cover microplates with TopSeal-A Adhesive Sealing
Films to reduce evaporation during incubation. Microplates can be read with the TopSeal-A Film. • Alpha signal is detected using an EnVision® Multilabel Reader equipped with the Alpha option using the following
• AlphaLISA® signal will vary with temperature and incubation time. For consistent results, identical incubation times and temperature should be used for each plate.
Materials Required but Not Provided
Item Suggested source Catalog # Size
5 rabbit IgG antibodies against the target protein of choice, in order to select one for the optimal assay
- - -
5 mouse IgG antibodies against the target protein of choice, in order to select one for the optimal assay
- - -
HardShell PCR Plate, 96 wells, Blue PerkinElmer Inc. 6008870 50/box
HardShell PCR Plate, 384 wells, Blue PerkinElmer Inc. 6008910 50/box
TOPSEAL-B FOR PCR PLATE PerkinElmer Inc. 6050174 100/box
HBSS (1x) Hank’s Balanced Salt Solution (with CaCl2 and MgCl2) Gibco 14025-050 500 mL
Optiplate-384, White Opaque assay plate (1) PerkinElmer Inc. 6007290 50/box
TopSeal-A 384, clear adhesive sealing film PerkinElmer Inc. 6050185 100/box
Veriti 96 Well Thermal Cycler : 96-well format PCR Machine, with 6 temperature zones (6 x 2 columns), useful for performing target melting curves
Applied Biosystems
- -
ProFlex PCR System : Can accommodate 2 x 384-well plates at once; same temperature for all the plate
Applied Biosystems
- -
Microplate shaking table - - -
Envision®, Ensight™ or Victor® Nivo™ Alpha-reader PerkinElmer Inc. - -
(1) Plates used for the immunoassay; (2) Same as (1) but optimal if cross-talk needs to be reduced; For more assay plates options, please go to www.perkinelmer.com/microplates
Buffer Preparation and Subsequent Storage Conditions
2X Lysis Buffers
Dilute each 5X Lysis Buffer in MilliQ water to a final concentration of 2X.
For example: for 5 mL of 2X Lysis Buffer, add: 2 mL of 5X Lysis Buffer to 3 mL MilliQ water. Excess 2X Lysis Buffer should be discarded.
1X AlphaLISA Immunoassay Buffer Add 10 mL of 10X AlphaLISA immunoassay Buffer to 90 mL MilliQ water.
5X Acceptor Mix
Add 50 µL of 5 mg/mL AlphaLISA Anti-rabbit IgG Acceptor beads to 4950 µl of 1X AlphaLISA Immunoassay Buffer to make a 5X beads suspension (50 µg/mL), in order to reach a final concentration of 10 µg/mL
5X Donor Mix
Add 200 µL of 5 mg/mL anti mouse IgG Donor beads to 4800 µL of 1X AlphaLISA Immunoassay Buffer to make a 5X beads suspension (200 µg/mL), in order to reach a final concentration of 40 µg/mL
All buffer and beads dilutions should be prepared the same day as the experiment is run and used immediately.
Standard Assay Volume Conditions
The table below describes the volumes of each reagent type to be engaged according to the standard protocol of the kit. These calculations do not include excess reagents to account for losses during transfer of solutions or dead volumes.
Assay Procedure for the development of an Alpha CETSA® Immunoassay
The development of an Alpha CETSA® assay involves selecting an antibody pair for the detection of the soluble target protein of interest, optimizing the immunoassay and validating the assay with reference compounds know to bind to the target.
A. Antibody Pair Selection
Antibodies selection
The first step in the development of an Alpha CETSA® assay is the selection of an antibody pair for the detection of the target protein of interest. For that purpose, several antibodies are selected and all possible antibody pairs are tested in a multiple matrix experiment (Fig. 3).
We recommend selecting at least 5 rabbit and 5 mouse antibodies, as the quality (affinity, stability, specificity) of antibodies is the primary determinant for the sensitivity and quality of an immunoassay. The beads included in the kit recognize the Fc part of rabbit and mouse IgGs, and therefore attention must be paid that the antibody class is IgG if working with monoclonal antibodies (i.e. not IgA, IgM, etc…). Polyclonal antibodies can be selected as well, as they are expected to contain IgGs. In that case we recommend that the polyclonal antibodies be affinity purified using the antigen used for immunization.
It is a good idea to carefully select antibodies targeting different epitopes on the target protein, to cover a variety of epitopes distributed at different positions on the target protein, in order to maximize the chances that there will be some of the epitopes that will be the most sensitive to the heat treatment of the sample, and to the stabilization of the protein by small molecule compound binding. This is particularly important when working with monoclonal antibodies, as they each recognize a single epitope on the protein.
Antibody pair selection
• 5 rabbit X 5 mouse antibodies• Monoclonal (IgG) or polyclonal• Variety of epitope coverage• Test all possible antibody pairs on lysates from cells heated at 37°C vs 70°C.
Immunoassay optimization
• Antibodies concentration• Assay orientation• Order of addition• Incubation times• Others
CETSA Validation
• Cell density & Lysis buffer Optimization• Melting curve• Thermal shift by reference compounds → selection of a single temperature for compound testing• Reference compounds dose-response
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Figure 3. Principle of Antibody Pair Selection to setup an Alpha CETSA® ToolBox assay. 5 rabbit and 5 mouse antibodies are selected, ideally covering a variety of epitopes on the target protein of interest. A cell type expressing the target protein of interest is selected, and a disrupted cell suspension is prepared by 3 freeze-thaw cycles. Some aliquots of this disrupted cell suspension are heat-inactivated (70°C, background signal), while others are kept intact (37°C, maximum potential signal). All possible antibody pairs are then tested using these positive and negative samples with each antibody at 3 concentrations and including a “no antibody” condition to detect if a particular antibody would generate elevated background signal by itself. This makes a total of 25 antibody pairs to test (i.e. 25 plates, each containing 64 wells to test, making a total of 1600 datapoints). The antibody pair & concentration yielding the best Alpha signal to background is selected for further assay optimization.
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Sample preparation for Antibody pair screening
To be able to screen the antibody pairs and identify the best one, you will need some samples that contain the target protein of interest. Select a cellular model where the target of interest is known to be present. Validation by other methods, such as Western Blotting or ELISA, of the presence of the target, and selection amongst a few cellular models of one expressing high levels of the target is always a good idea as it may avoid spending time trying to develop an assay using samples where the target is just not present, or not present in sufficient quantities.
In order to avoid any issue that may arise from poor target release from the cells, and that may require further cell lysis optimization, we recommend at this stage to work with cells disrupted by a freeze-thaw process:
Prepare a bulk suspension of cells disrupted by 3 freeze-thaw cycles:
• Culture the cells until 70% confluency using their optimal culture medium and tissue culture surface treatment. 150 or 225 cm² tissue culture plates or 175 cm² T-Flasks can be used at this stage.
• Wash the cells with 5 mL of TrypLE (ThermoFisher 12563-029). • Harvest the cells in 5 mL TrypLE for a 150 cm² cell culture flask. • Collect the remainder of the cells from the plates using Hank’s Balanced salt Solution (HBSS, ThermoFisher
14025-050). • Pellet the cells by centrifugation at 300 rpm for 3 min. • Wash the cells twice in HBSS. • Count the cells (e.g. by trypan blue exclusion). • Resuspend the cells in HBSS at a density of 10 million cells/mL.
Note: The cell density of 10 Million cells/mL is a recommended starting point, but if you know that your target protein of interest is expressed at very high levels in the cell type selected, you may start with a lower cell density, such as 2 or 5 million cells/mL, in order to avoid hooking the immunoassay at the stage of antibody pair screening.
• Aliquot the cells by 1 mL of cells suspension in 2 mL tubes. • Freeze the tubes by plunging them in liquid nitrogen until the tube content is completely frozen.
Note: make sure you comply with standard lab safety procedures when working with liquid nitrogen to avoid any burning by liquid nitrogen. In particular wear protective glasses and gloves and close the tubes tightly to avoid any risk of tube explosion consecutive to liquid nitrogen entering in the tubes.
• Thaw the tubes in a water bath at 22°C until the tube content is completely thawed. • Repeat the freeze-thaw cycle another 2 times. • Centrifuge the tubes for 15 min in an Eppendorf tube centrifuge at the highest sped (i.e. 13 000 to 17 850 rpm)
at 4°C to clear the suspension from cell debris. Note: Omitting the centrifugation step may result in high levels of background signal.
• Transfer the supernatant in another tube and use immediately. Note: Do not add cell lysis buffer before the thermal treatment (70°C, see below) of the sample, as detergents present in the cell lysis buffers may interfere with the thermal denaturation profile of the target protein.
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As the goal of the antibody pair screening is to identify a pair generating the best signal when the target protein has not been heat-challenged, and at the same time generating the lowest background signal when the target has been heat-challenged, aliquots of the disrupted cells preparation are heat-challenged, and other aliquots are kept intact. Most proteins are heat-inactivated at 70°C. For that reason some aliquots of the sample are heated for 3 min either at 37°C (positive control, where the intact target protein is expected to be present) and other aliquots of the sample are heated at 70°C (negative control, where the target protein is expected to be heat inactivated).
Heat inactivate an aliquot of the disrupted cells suspension:
• In a PCR plate, add 20 µL/well of disrupted cell suspension prepared above. Note: up to 60 µL/well of disrupted cell suspension can be engaged per well of the PCR plate, as this allows minimizing the number of wells to handle. Do not exceed 60µL/well as it may result in incomplete heating of the sample, due to the thermal inertia of the sample and to the time it takes for heat transfer.
• Pre-heat the PCR thermocycler to the either 37°C or 70°C. • Transfer the plate into the PCR thermocycler and heat the samples for 3 minutes. • Remove the plate from the PCR thermocycler and allow it to cool down to 4°C on ice or use the thermocycler for
fastest possible cooling. Incubate for at least 3 minutes. • Add 20 µL of freshly prepared 2X CETSA Cell Lysis Buffer. Agitate on a plate shaking table (350 to 700 rpm) for
10 minutes at room temperature. Note: the selection from the CETSA Cell Lysis Buffer 1 to 5 is part of the optimization of an Alpha CETSA® assay, however if wanting to select only one CETSA Cell Lysis Buffer at this stage, we recommend using here either CETSA Cell Lysis Buffer 1 or 2. See the table on page 7 for guidance.
• Pipet 20 µL up and down three times to ensure homogeneity of the cell lysate solution. • Transfer 10 μL of the lysate to a 384-well Optiplate™ or AlphaPlate™ for the immunoassay. This corresponds to
50 000 cells/well.
Note: A few proteins are exceptionally not heat inactivated at 70°C and will require a higher temperature to be heat inactivated. From the analysis of a wide repertoire of 7000 proteins by mass spectrometry (CETSA®-MS), Pelago Bioscience may already have information available about the expected heat-inactivation profile of your protein of interest.
Include a recombinant protein control if available:
If a recombinant protein is available, it can be useful to include it in the antibody pair screening as it will provide a control that the signal detected is well due to the presence of the protein of interest. However please note that in our experience heat inactivation of recombinant proteins is often not possible, as accompanying carrier proteins, such as BSA, used to stabilize the purified target protein of interest, can be present in relatively high quantities and lead to massive aggregation of the sample upon heating. Beside the issue that carrier proteins may cause, the thermal inactivation profile of a purified, isolated protein may not be representative of the in vivo or cellular situation, where partner proteins and different cofactors interacting with the target protein, and more broadly the cellular context, may lead to a different thermal shift profile, and to a different shift by reference compounds.
If wanting to select a single recombinant protein concentration as a control you may engage a concentration of 1 ng/mL (10 µL/well) as this is usually in the range detected when screening antibody pairs. However be aware that if the assay is very sensitive, using less recombinant protein may be necessary to avoid a hooking effect.
The Alpha CETSA® Assay is primarily intended to detect changes in soluble analyte following compound treatment. Therefore, conversion of Alpha signal into analyte concentration value is normally not done in a CETSA® assay.
However, performing a standard curve using a recombinant protein or some validated reference frozen control lysates is still useful for controlling the AlphaLISA immunoassay performance.
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Antibody pair screening
Test all possible antibody pairs, using 3 antibody concentrations (0.3 nM, 1 nM and 3 nM, corresponding to 48, 160 and 480 ng/mL) of each antibody, as well as a “no antibody” control, and 10 µL/well of the cell lysate, that has been heated either at 37°C (positive control) or at 70°C (background signal). We recommend testing each condition in duplicate or triplicate.
To the plate containing 10 µL of cell lysate/well:
• Add 10 μL of a 5X rabbit antibody solution (prepare 1.5 nM, 5 nM and 15 nM solutions to get final 0.3 nM, 1 nM and 3 nM in the assay) prepared in 1X AlphaLISA ImmunoAssay Buffer.
• Add 10 µL of the mouse antibody solution (prepare 1.5 nM, 5 nM and 15 nM solutions to get final 0.3 nM, 1 nM and 3 nM in the assay) prepared in 1X AlphaLISA ImmunoAssay Buffer. Seal plate with Topseal-A adhesive film and incubate for 1 hour at room temperature Note: When working with purified polyclonal antibodies, the supplier usually provides the antibody concentration. As a reference, an antibody at 1 mg/mL has a concentration of 6250 nM, assuming an average molecular weight of 160 000 g/mol. We do not recommend working with non-purified polyclonal antibodies as the target-specific titer may be quite low.
• Add 10 μL of 5X Acceptor Mix (50 µg/mL) to the wells. Seal plate with Topseal-A adhesive film and incubate for 1 hour at room temperature.
• Add 20 μL of 5X Donor Mix (200 µg/mL) to the wells under subdued light. Seal plate with Topseal-A adhesive film, and protect the plate from light. Incubate for 1 hour at room temperature in the dark. Note: Longer incubation may give greater sensitivity. Plates can be incubated overnight if required.
• Read plate on an Alpha Technology-compatible plate reader, using standard Alpha settings. • Select the best antibody pair or up to the 3 best antibody pairs, based on the maximal signal obtained with the 37°C
sample, and on the maximal signal to background (37°C sample value / 70°C sample value) obtained.
Note: In our experience, testing each antibody at 0.3/1/3 nM against each antibody at 0.3/1/3 nM is worth the investment, as if not doing so, some interesting antibody pairs may be completely missed.
Note: Pairs of antibodies targeting the same exact epitope on the protein may be skipped from the screening process.
Note: If no or weak signals are obtained at this stage, consider repeating the test using dilutions of cell lysates, e.g. 3X and 10X diluted in 1X CETSA Cell Lysis Buffer, as the initial weak signals may be due to hooking of the immunoassay by saturating target protein concentrations.
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B. Immunoassay Optimization
Once the optimal antibody pair will have been selected, we recommend spending some time to optimize the Alpha Immunoassay, as it can result in better sensitivity and more convenient protocols. These factors are important as they allow to minimize the quantity of cellular material that needs to be engaged in the assay, to get better data quality and hence to avoid spending time trying to confirm hits that were not true hits, and to enable less hands-on assay time or easier automation of the assay.
Such immunoassay optimization can also be performed on the 3 best antibody pairs, in order to select the best from the best and get the very best final assay possible.
There are multiple parameters that may be optimized, which include but are not limited to:
- Antibody concentration optimization - Assay orientation optimization (reverse orientation testing) - Order of addition and number of addition steps optimization - Incubation time optimization - Assay Buffer optimization - Others
For a complete list of potential Alpha assay optimization points, please refer to the “User’s Guide to Alpha Assays Protein:Protein Interaction” (which despite its name contain many advices worth for the development of other types of Alpha assays) to the “AlphaLISA Assay Development Guide” and to “A Practical Guide to working with AlphaScreen”.
We recommend that antibody concentration optimization be part of any Alpha CETSA® Toolbox assay optimization, and advise to consider optimizing the other parameters too unless the characteristics of the assay obtained after this stage would already be satisfactory enough for the intended use of the assay (e.g. if testing only one or a few molecules and the assay window already allows to detect clear signals it may not be worth spending too much time on optimizing the assay).
Antibody concentration optimization
For each antibody pair you want to optimize, we recommend testing 0.1 nM, 0.3 nM, 1 nM, 3 nM and 10 nM of each antibody concentration, for the detection of 37°C/70°C cell lysates, undiluted and 10X diluted (Fig. 4). From there, the assay setting providing the maximal signal with the 37°C sample, and the maximal signal to background (37°C sample value / 70°C sample value) will be selected.
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Figure 4. Antibody concentration optimization. Each antibody is tested against the other antibody at 5 concentrations, in order to identify the optimal concentrations. This is done on undiluted cell lysates, and on 10X diluted lysates to be sure not to miss the optimal combination in case the undiluted sample concentrations would be too high and hook the assay.
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Reverse orientation testing
The CETSA-TBX2-A kits capture the rabbit antibody on the Acceptor beads side, and the mouse antibody on the Donor beads side, while the CETSA-TBX1-A kits capture the mouse antibody on the Acceptor beads side, and the rabbit antibody on the Donor beads side (Fig. 5).
A. CETSA-TBX2-A kits Assay configuration: Anti-mouse IgG Donor X Anti-rabbit IgG Acceptor Beads
B. CETSA-TBX1-A kits Assay configuration: Anti-rabbit IgG Donor X Anti-mouse IgG Acceptor Beads
Figure 5. Comparison of the configuration of the CETSA-TBX2-A kits (A) and of the CETSA-TBX1-A kits (B).
The anti-rabbit IgG antibody present on the anti-rabbit IgG Acceptor Beads of the CETSA-TBX2-A kits is the same as the anti-rabbit IgG antibody present on the anti-rabbit IgG Donor Beads of the CETSA-TBX1-A kits; and similarly the anti-mouse IgG antibody present on the anti-mouse IgG Donor Beads of the CETSA-TBX2-A kits is the same as the anti- mouse IgG antibody present on the anti- mouse IgG Acceptor Beads of the CETSA-TBX1-A kits.
However in practice the performance of one or the other orientation of the assay (i.e. rabbit antibody on the Acceptor or on the Donor side) may differ, and for that reason we recommend, when optimizing an Alpha CETSA® assay, to compare the performance of the CETSA-TBX2-A kits with those of the CETSA-TBX1-A kits. One of the reasons that may be responsible for the difference in performance of the two possible orientations can be that the binding capacity differs between the Donor and Acceptor beads, due to differences in the antibody conjugation protocols used to manufacture both types of beads (i.e. a higher antibody binding capacity is expected for the Donor beads), but other factors such as the order of addition used can also have an impact.
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Other parameters for assay optimization
Incubation times:
One hour of incubation may not always be sufficient to reach equilibrium, and hence the maximal level of signal. For that reason, if the assay window needs to be increased, incubation times can be extended, up to overnight for the last incubation. On the other hand, longer incubations may sometimes lead to an increase of non-specific binding of the antibodies, or to reagents aggregation, increasing the level of background.
The excitation of Alpha Donor beads somehow “bleaches” the reagents, meaning that in theory if the Alpha signal is read from a well one time, the next time the same well will be read again the level of Alpha signal is expected to decrease. This effect is significant when working in 1536-well format, and visible when working in 384-well format, but in practice it is useful to read the same 96- or 384-well plate several times (e.g. after 1h, 4h and overnight incubation) when developing an assay to get a first idea of how the signal and background evolve over time (and keeping in mind that due to bleaching the level of counts for reading 2 and 3 are not absolute).
Assay Buffer components:
Some proteins are less stable than others, more sticky than others, and this is true for antibodies too. For that reason, it may happen that the AlphaLISA ImmunoAssay Buffer (PerkinElmer AL000) is not the optimal one, and that more blocking agents are needed to prevent the apparition of a too high level of background signal. For that purpose, Hi-Block Assay Buffer (PerkinElmer AL004), which contains additional 0.5% BSA and 0.5% gelatin in order to prevent further non-specific binding events is available and can be purchased separately for testing.
If the assay performance is still not satisfactory, other variations in the assay buffer composition, like trying other types of detergents, divalent cations, pH, or target-specific buffer components may be tried.
Number of addition steps:
The protocol provided here is the “extended”, “long-play” version of the assay as à priori this is where to start to be sure to get the maximal performance of the assay. However, to reduce the number of pipetting steps, combining some of the additions may be tried and compare to the “extended” version of the protocol. For example the two antibodies can be pooled and added in a single addition step, and the two types of beads can be pooled and added in a single addition step. It may even be possible to pool all the antibodies and the beads, and to have a single reagents addition step. The key is to compare, validate and select a configuration that meets the requirements of the assay performance.
CETSA Cell Lysis Buffer:
Up to this point, the Alpha CETSA® immunoassay has been developed using disrupted cells, to make sure that the target protein is accessible to antibodies used in the immunoassay. However the main interest of a CETSA® assay is the ability to run the assay on live cells, in order to get the most significant information. In that situation, live cells will be incubated with the compound first, then heat-challenged, and then only lysed. PerkinElmer is making available 5 different CETSA Cell Lysis Buffers (see below), and the selection of the best assay buffer is an important part of the optimization of a CETSA® assay.
Other parameters
Other parameters, including varying the sample volume and the final assay volume, the incubation temperature, testing different assay settings, for example, directly conjugating antibodies to the Alpha beads, varying beads concentrations. As stated above, please refer to the Alpha technology guides for the detail of Alpha assay optimizations.
When wanting to demonstrate the specificity of an immunoassay, using competitors such as peptides that have been used to raise the antibodies is always a very useful approach.
At this stage, you have selected an immunoassay configuration that allows you to detect your target protein of choice remaining soluble after thermal treatment, now it is time to move to the next stage and to validate your CETSA® assay using reference compounds, using the protocols below.
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Alpha CETSA® Assay Protocols
The most relevant CETSA® assay is performed on living cells, as data obtained in such protocols reflect the ability of the compound to reach and bind the target in a close-to-physiological situation. However, there is a possibility to run CETSA® assays on cells disrupted by freeze-thawing prior to being placed in the presence of the test compounds. This may provide valuable information about the ability of the compound to engage the target, independently of its permeability and metabolization properties by a living cell.
A. Intact Cells CETSA® assay
Cell Preparation
1. Recover cells directly (suspension cells) or by trypsination (adherent cells) from T-Flasks, Petri Dishes or Cell Factories (or any other culture method in place for the cell type used). Then wash the cells in HBSS to remove trypsin and resuspend cells in HBSS. A common density to start with is 1 to 2 million cells/mL, but cell density is part of the initial optimization of CETSA® assays and this can vary from cell type to cell type and according to the target to be detected.
Cell Treatment
2. In a PCR plate, add 10 µL/well of 2X concentrated test compound diluted in HBSS. When performing concentration-response testing, it is recommended to do a first dilution in 100% DMSO and then further dilute each compound concentration in HBSS, in order to keep the final DMSO concentration the same in all samples. Note: DMSO concentration should be kept at maximum 0.1% final on cells in order to avoid toxic effects.
3. Add 10 µL/well of cells resuspended in HBSS. 4. Incubate at 37°C / 5% CO2 (in a cell culture incubator) for 60 min.
Note: 30 minutes is often sufficient for intracellular targets, but the optimal incubation time may vary according to the target and cell type used. Note: Some targets are unstable and incubation at lower temperatures than 37°C may be required.
5. Pre-heat the PCR thermocycler to the selected temperature. Note: For measuring a target melting curve, a thermocycler with variable temperature zones is very useful at this stage. - Please pay attention that “gradient PCR machines” do not provide a precise control of the temperature over different zones and therefore are not recommended for running melting curves as this could introduce a bias in the true melting temperature calculation. - A standard melting curve typically includes 12 different temperatures ranging between 37-63°C. When one temperature for CETSA® screening applications has been selected, a thermocycler with a single temperature zone can be used. Important Note: The heating of the cover should be inactivated, else this would result in higher than desired sample temperatures and inaccuracy of melting temperature calculation.
6. Transfer the plate into the PCR thermocycler and heat the samples for 3 minutes. Note: Heating time is an important parameter, and it is important to strictly control it. Using shorter or longer heating times may result in a different concentration-response profile. In particular, compounds with different retention times by the target (off-rates) are expected to be impacted differently by changes in plate heating time.
7. Remove the plate from the thermocycler and allow it to cool down to 4°C on ice or use the thermocycler for fastest possible cooling. Incubate for at least 3 minutes.
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Lysate Preparation 8. Add 20 µL of freshly prepared 2X CETSA Cell Lysis Buffer. Agitate on a plate shaking table (350 to 700 rpm) for 30
minutes at room temperature. Note: An additional lysis step of flash freezing in liquid nitrogen and thawing at 20°C using the thermocycler can be implemented at this stage if desired as it may in some cases improve the day-to-day and/or well-to-well signal variability. Note: make sure you comply with standard lab safety procedures when working with liquid nitrogen to avoid any burning by liquid nitrogen. In particular wear protective glasses and gloves.
9. Pipet 20 µL up and down three times to ensure homogeneity of the cell lysate solution. 10. Transfer 10 μL of the lysate to a 384-well Optiplate™ or AlphaPlate™ microplate for the immunoassay.
Alpha CETSA® Assay
11. Add 10 μL of the mouse antibody. 12. Add 10 µL of the rabbit antibody. Seal plate with Topseal-A adhesive film and incubate for 1 hour at room
temperature Note: Once the optimal antibody concentration will have been determined, the two antibodies can be combined in a pre-mix, and 20 µL of this pre-mix added instead of 2X10 µL in order to minimize the number of addition steps.
13. Add 10 μL of 5X Acceptor Mix to the wells. Seal plate with Topseal-A adhesive film and incubate for 1 hour at room temperature.
14. Add 20 μL of 5X Donor Mix to the wells under subdued light. Seal plate with Topseal-A adhesive film, and protect the plate from light. Incubate for 1 hour at room temperature in the dark. Note: Longer incubation may give greater sensitivity. Plates can be incubated overnight if required.
15. Read plate on an Alpha Technology-compatible plate reader, using standard Alpha settings.
Read Settings: Alpha signal is detected using an EnVision Multilabel Reader equipped with the Alpha option using the following settings: Total Measurement Time: 550 ms, Laser 680 nm Excitation Time: 180 ms, Mirror: 640as (Barcode# 444), Emission Filter: Wavelength 570nm, bandwidth: 100nm, Transmittance 75%, (Barcode# 244).
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B. Disrupted Cells CETSA® assay
Cell Preparation
1. Recover cells directly (suspension cells) or by trypsinization (adherent cells) from T-Flasks, Petri Dishes or Cell Factories (or any other culture method in place for the cell type used). Then wash the cells in HBSS to remove trypsin and resuspend cells in HBSS. A common density to start with is 1 to 2 million cells/mL, but cell density is part of the initial optimization of CETSA® assays and this can vary from cell type to cell type and according to the target to be detected.
Cell disruption
2. Dispense the cell suspension in 1 mL to 10 mL tubes. 3. Freeze the cells by plunging the tubes into liquid nitrogen, until the tube content is completely frozen.
Note: make sure you comply with standard lab safety procedures when working with liquid nitrogen to avoid any burning by liquid nitrogen. In particular wear protective glasses and gloves and close the tubes tightly to avoid any risk of tube explosion consecutive to liquid nitrogen entering in the tubes.
4. Thaw cell suspensions by plunging the tubes in a water bath at 20°C, until the tube content is completely thawed. 5. Repeat the freeze thawing three times. 6. Optional: Centrifuge the tubes for 15 min at 20 000xg to remove cell debris and transfer the supernatant into a new
tube, and use the disrupted cells suspension immediately Note 1: In some cases using non-centrifuged disrupted cell suspension can lead to elevated background. In such case, the suspension can be cleared by centrifugation, as this usually decreases the background signal. Note 2: Depending on the target and cell type used, there may be a possibility to store the disrupted cell solution at -80°C for later use. However, some targets may degrade over time when stored in such conditions, and this needs to be validated on a case-by-case basis.
Disrupted cells Treatment
7. In a PCR plate, add 10 µL/well of 2X concentrated test compound diluted in HBSS. When performing concentration-response testing, it is recommended to do a first dilution in 100% DMSO and then further dilute each compound concentration in HBSS, in order to keep the final DMSO concentration the same.
8. Add 10 µL/well of disrupted cell suspension prepared above. 9. Incubate at 37°C / 5% CO2 (in a cell culture incubator) for 30 minutes. 10. Pre-heat the PCR thermocycler to the selected temperature. 11. Transfer the plate into the PCR thermocycler and heat the samples for 3 minutes.
Note: Heating time is an important parameter, and it is important to strictly control it. Using shorter or longer heating times may result in a different concentration-response profile. In particular, compounds with different retention times by the target (off-rates) are expected to be impacted differently by changes in plate heating time.
12. Remove the plate from the PCR thermocycler and allow it to cool down to 4°C on ice or use the thermocycler for fastest possible cooling. Incubate for at least 3 minutes.
13. To keep the same buffer conditions as when working with intact cells, add 20 µL of freshly prepared 2X CETSA Cell Lysis Buffer. Agitate on a plate shaking table (350 to 700 rpm) for 10 minutes at room temperature.
14. Pipet 20 µL up and down three times to ensure homogeneity of the cell lysate solution. 15. Transfer 10 μL of the lysate to a 384-well Optiplate™ or AlphaPlate™ microplate for the immunoassay.
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Alpha CETSA® Assay 16. Add 10 μL of the mouse antibody. 17. Add 10 µL of the rabbit antibody. Seal plate with Topseal-A adhesive film and incubate for 1 hour at room
temperature Note: Once the optimal antibody concentration will have been determined, the two antibodies can be combined in a pre-mix, and 20 µL of this pre-mix added instead of 2X10 µL in order to minimize the number of addition steps.
18. Add 10 μL of 5X Acceptor Mix to the wells. Seal plate with Topseal-A adhesive film and incubate for 1 hour at room temperature.
19. Add 20 μL of 5X Donor Mix to the wells under subdued light. Seal plate with Topseal-A adhesive film, and protect the plate from light. Incubate for 1 hour at room temperature in the dark. Note: Longer incubation may give greater sensitivity. Plates can be incubated overnight if required.
20. Read plate on an Alpha Technology-compatible plate reader, using standard Alpha settings.
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Supplementary Buffers
If using the standard protocol, sufficient amounts of buffers are provided in the kit. However if the standard protocol is modified, more buffers may be needed. In this case, additional buffers can be ordered using the following catalog numbers:
CETSA® Cell Lysis Buffer 2 PerkinElmer Inc. CETSA- BUF2-100mL 100mL CETSA® Cell Lysis Buffer 3 PerkinElmer Inc. CETSA- BUF3-100mL 100mL
CETSA® Cell Lysis Buffer 4 PerkinElmer Inc. CETSA- BUF4-100mL 100mL
CETSA® Cell Lysis Buffer 5 PerkinElmer Inc. CETSA- BUF5-100mL 100mL
Useful Links For FAQ and troubleshooting, please go to: www.perkinelmer.com/CETSAFAQ For a complete list of Alpha CETSA® kits, please go to: http://www.perkinelmer.com/category/alpha-CETSA-kits or https://www.pelagobio.com/ For technical support please go to: www.perkinelmer.com/ASK Recommended CETSA® Reading Martinez-Molina. D. (2013). Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay. Science 341:84-87. https://science.sciencemag.org/content/341/6141/84 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. https://dx.doi.org/10.1021/acs.biochem.8b01057 Shaw J, Dale I, Hemsley P, Leach L, Dekki N, Orme JP, Talbot V, Narvaez AJ, Bista M, Martinez-Molina D, Dabrowski M, Main MJ, Gianni D. (2019) Positioning High-Throughput CETSA in Early Drug Discovery through Screening against B-Raf and PARP1. SLAS Discovery 24:121-132. https://journals.sagepub.com/doi/10.1177/2472555218813332 More complete list of publications available at www.pelagobio.com/Publications
