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We offer a comprehensive portfolio of products and services to investigate the major druggable target classes: kinasespathways, GPCRshERG, and nuclear receptors. Our offerings span from high-quality reagents for basic research and assay development to validated biochemical and cell-based assays. We also offer world-class SelectScreen profiling and screening services with project sizes ranging from one compound/one target to hundreds of thousands of compounds.

Please take a look at all our offerings here.

We specialize in ratiometric fluorescence-based assays including TR-FRET, FRET and fluorescence polarization (FP). Ratiometric assays reduce the effects of well-to-well and day-to-day variations and improve the noise characteristics of the assay as determined by the Z’-factor so that inhibition can be more readily distinguished from noise.

The first step is to determine if your microplate reader has the capability of doing TR-FRET, FRET or fluorescence polarization (FP). Please visit our instrument compatibility portal to see our instrument setup guides.

  • TR-FRET: The instrument must have the capability of a time-delayed detection, typically 100 µsec. Filter requirements for TR-FRET are very exacting, please see our instrument guides here.
  • FRET: Most fluorescent microplate readers can do FRET as these are simple fluorescent assays except that there is one excitation wavelength and two emission wavelengths. We typically use coumarin and fluorescein as the FRET pair and these filters are commonly available on most instruments. If your plate reader can only read one emission wavelength at a time, then the plate can be read twice.
  • Fluorescence polarization: fluorescence polarization is a specialized technique that must be built into the microplate reader. This is not the same as a fluorescence intensity reading. Check with your instrument manufacturer to see if fluorescence polarization was included on your instrument.

The single most common reason that a ratiometric fluorescent assay fails is due to instrument setup and not due to the components of the assay. Please refer to our instrument setup guides in our instrument compatibility portal or contact Drug Discovery Technical Support at

These assays require the use of a microplate reader; most ion channel assays also require the use of an automated dispensing system. Please refer to our instrument setup guides in our instrument compatibility portal. If your instrument is not listed there, please contact Drug Discovery Technical Support at

Filter specifications will list the wavelength that the filter is centered on as well as the upper and lower limits of wavelength of light that the filter will pass. If a filter is listed at 520/25 nm, this means that the filter is centered at 520 nm and light passes between 520 +/- 12.5 nm, or 507.5 to 532.5 nm.

Initial investigations are typically done with biochemical assays with cell-based assays as follow-up.

It is quite common to test a single concentration of the test compounds; 10 µM is a typical concentration. Use at least duplicates.

  • Concentrations above 10 µM are more prone to either precipitation or fluorescent interference, even in ratiometric fluorescent assays.
  • Sometimes 3 concentrations such at 10 µM, 1 µM and 100 nM are used. Please note that this will not provide IC50 information.

When controls are used with the assay, it will be possible to determine the percent inhibition of the test compound. By definition, 50% inhibition is the IC50 of the compound.

  • So for example, if the test compound at 10 µM shows 50% inhibition, then the IC50 is 10 µM.

Typically, with a Hill Slope of 1, a compound that shows 100% inhibition will have an IC50 two logs below 10 µM.

The concentration in mg/mL is lot-specific and is listed in the Certificate of Analysis (COA). The volume can be calculated from the concentration knowing the amount purchased. Please be sure to briefly spin down the product to remove material that may be trapped in the cap.

For an acceptable inhibition curve, a minimum ten point, 3-fold dilution of the inhibitor or compound should be used. For example, if the initial maximum concentration to be tested is 10 µM, make a dilution series of 10 µM, 3.3 µM, 1.1 µM, down to 0.5 nM.

For kinases, the LanthaScreen Eu Kinase Binding Assay can be used to determine on- or off- rates of compounds. For further information, please refer to the LanthaScreen Eu Kinase Binding Assay portal and to this application note.

Please note that the LanthaScreen Kinase Activity, Adapta Kinase Activity and Z’-Lyte Kinase Activity assays are all endpoint assays and not suitable for determination of kinetic factors such as initial velocity or substrate Km.

We do not offer any kinetic nuclear receptor assays.

Please go to the product page for your kinase and look at the Manuals tab, there you will find a Validation Packet for your kinase with the required information.

Please refer to the Z’-LYTE Screening Protocol and Assay Conditions for the following information:

  1. The approximate amount of kinase required
  2. If additives or changes are needed for Kinase Buffer A
  3. The ATP Km apparent value for the assay
  4. The identity of the control inhibitor and its IC50 value

Here is an example using ERB2 (HER2) kinase. You will see information that looks like this:


  • The 2X ERBB2 (HER2) / Tyr 06 mixture is prepared in 50 mM HEPES, pH 7.5, 0.01% BRIJ-35, 10 mM MnCl2, 1 mM EGTA, 2 mM DTT, 0.02% NaN3.
  • The final kinase reaction consists of 1.78 - 30.4 ng ERBB2 (HER2) and 2 µM Tyr 06 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 5 mM MgCl2, 5 mM MnCl2, 1 mM EGTA, 1 mM DTT, 0.01% NaN3.
  • After the 1 hour kinase reaction, 5 µL of a 1:128 dilution of Development Reagent A is added.

Please note that the ERBBE (HER2) kinase requires Mn2+ for activity, and hence the kinase is not prepared in 1X Kinase Buffer A (50 mM HEPES, pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA).

The amount of kinase to use will be similar to what we show here depending on the lot number of the kinase and the ATP level that you select. Note that the 1.78–30.4 ng is the amount of kinase in 10 µL, not a concentration. Do a small titration of the kinase to target 30% phosphorylation of the Z’-LYTE substrate. Your ng usage should be similar to what we show.

Here is the premise of the above outline from SelectScreen. This is a variation on the customer protocol.

  1. 4X compounds are prepared in 1X Kinase Buffer A after the serial dilution is done at 100X in 100% DMSO.
  2. (2X Kinase/2X Substrate) is prepared at 2X in the kinase buffer. For ERBB2, the kinase buffer can be made from scratch or by spiking a concentrated form of the additives into 1X Kinase Buffer A. For ERBB2 kinase, this means addition of Mn2+, DTT and NaN3. Also, please see options below*.
  3. 4X ATP is prepared in 1X Kinase Buffer A.

Add to the assay plate:

  1. 2.5 µL of 4X compound.
  2. 5 µL of 2X Kinase/2X Substrate
  3. 2.5 µL of 4X ATP to start the reaction

The buffer used to prepare the kinase substrate mixture is 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MnCl2, 1 mM EGTA, 2 mM DTT and 0.02% NaN3. *There are a couple of options.

  1. Make the buffer from scratch.
  2. Spike in the additives to 1X Kinase Buffer A as follows. Note that we make the buffer from scratch, so this is just a mathematical example.

To 1 mL of 1X Kinase Buffer A add:

i.     2 µL of 5,000 mM MnCl2

ii.     2 µL of 1,000 mM DTT

iii.     2 µL of 10% NaN3

Then use this buffer to prepare the 2X kinase/2X substrate mixture.

After 1 hour, perform the kinase development reaction. Use the dilution outlined in the Certificate of Analysis (COA) that can be found on the product page, for the lot number of the kit purchased.


The Adapta Universal Kinase Assay Kit can be used on a variety of lipid kinases and kinase-specific protocols can be found on the product page for your kinase target of interest.

Most of our kinases are derived from insect cells. If the kinase is not listed as “Inactive” in its description title, the kinase is sold as an active kinase. The Certificate of Analysis (COA) for the respective kinase, found on the product page, will indicate how the kinase was activated. This information can be found in the upper left hand corner of the COA. For example, it may state, “No special measures were taken to activate this kinase”, in which case the insect cells produced it as an active kinase. We do not specifically study the phosphorylation state of the kinase either by mass spectrometry or antibodies. We look for consistent activity against the substrate. Most recombinant kinases have a mixture of phosphorylation states.

  • Please refer to the protocol for your target.
  • Most biochemical assays are read after 1 hour.
  • Cell-based BLA reporter assays are read after either 5 or 16 hours.

Ultrapure water may be derived from water treatment systems, but the spigot or tubing attached to spigots can become contaminated and in turn can contaminate the water as it is dispensed.

Please make sure that the membrane/filters are changed.

Purchase the highest-grade HPLC water.

Mineral contamination in water:

The chelates used in TR-FRET assays are very sensitive to Fe3+ contamination. Fe3+ competes with the Tb3+ or Eu3+ for the chelate and leads to a diminished or no assay window.

Sequestering of either the kinase or nuclear receptor, substrate, test compound or any component used in the assay can occur with different sorts of plastics.

Use the assay plates recommend in the protocol if there is one.

For TR-FRET Assays, white plates of the same plastic can be substituted for the black plates and they do perform better on some instruments and for assays with small windows.

White plates cannot be used for fluorescence polarization (FP) experiments.

Once an assay has been established and is running smoothly, please feel free to compare the results with any assay plate that you use more regularly.

Optimize and test the instrument prior to performing the assay.

Test the instrument settings using reference fluorophores or standards.

Include a water blank of the same volume as the assay. Since a “water blank” is non-fluorescent, it provides information on instrument noise, electrical variances or other issues unrelated to the assay reagents.

Contact the instrument manufacturer to improve optimization. Optimizing the instrument may include adjusting the gain (distance of optics from sample) and the voltage on the photomultiplier tube (PMT; the detector).

We offer a FP One-Step Reference Kit to detect in green (Ex/Em = 488/535 nm) and red (Ex/Em = 525/590 nm) emission ranges, suitable for the PolarScreen Green and Red Assays. The alternative is to perform the Controls in the respective assay kit prior to performing the actual assay using test compounds.

For FP assays, plate readers must specifically have FP built in. Please see our instrument compatibility portal.

Instrumentation: For a list of instruments, please visitourinstrument compatibility portal. Make sure that you have an instrument capable of doing bottom reads. Clear bottom plates are used and only those cells at the bottom of the assay plate contribute to the assay.

Media: The type of FBS specified in the protocol is critical, both for cell survival upon thaw and assay performance. In general, do not substitute media or supplements.

Storage: Proper storage of the BLA reporter cell lines is critical. Cells are shipped overnight at -80degrees C and must be either stored in liquid nitrogen or placed immediately into culture. Storage at -80degrees C is not a suitable substitute for liquid nitrogenstorage.

This is probably not a good idea. There can be differences in culture or assay requirements between cell lines of the same cellular background (cell type). This is because the cell lines were isolated by FACS and developed from a single cell. For example, some BLA HEK293T cell lines require poly-D-Lysine plates and other cell lines require the standard tissue culture treated plates. Please follow the protocol for each cell line.

Shortly after receipt of the cells, we recommend making an early passage bank. The cells can be kept in culture for typically 2 months, with passaging twice per week.

Adapta Universal Kinase Assay

In an assay designed to identify ATP-competitive inhibitors, the assay is typically run using an ATP concentration that is at or below the ATP Km value for the kinase of interest.

Because of the way Adapta assays are configured, it is very difficult to determine an ATP Km value using Adapta assays, and the ATP Km should be determined using another assay format (such as a traditional radiometric assay).

For many kinases where we have specifically developed an Adapta assay, we have determined the ATP Km in a radiometric assay. This data can be accessed in the Adapta Assay reactivity table.

It is important that the concentration of substrate present in the reaction is sufficient so it will not be limiting in the kinase reaction.

For example, if a kinase is assayed using 100 µM ATP, and the assay is performed such that data will be taken from the first 10% conversion of ATP to ADP (i.e., formation of 10 µM ADP), then 10 µM substrate will need to be converted to phosphorylated product. To ensure that the concentration of substrate will not limit the rate of the reaction over the course of this conversion, the substrate will need to be present at a concentration at least 5- to 10-fold above the concentration of ADP produced, (i.e., present at least at half the concentration of ATP that is used in the reaction).

Additionally, increasing the substrate concentration above this can often be beneficial by increasing the rate of the kinase reaction, therefore allowing less kinase to be used in the assay. In general, the benefits of using more substrate need to be weighed against practical matters such as substrate availability and cost.

In general, as the concentration of ATP goes up, more ADP Tracer is required.

The recommended Tracer concentrations for different ATP levels are stated on the antibody Certificate of Analysis that is provided with the kit (when using Kinase Buffer A, Cat. No. PV3189). The concentration of ADP Tracer can change with the manufacturing lot of antibody used. 

Optimization of the concentration of ADP Tracer is described in the Adapta Universal Kinase Assay User Guide 

There is some affinity of ATP toward the ADP antibody.

The reason that the optimal concentration of Tracer changes as the concentration of ATP changes is because the antibody used in the Adapta assay has a slight affinity for ATP. At higher concentrations of ATP, ATP itself can compete with the Tracer for binding to the antibody. As a consequence, more Tracer is required in the assay in order to saturate 50% of the antibody with Tracer, since the Tracer must “compete off” this bound ATP.

The Adapta assay is what can generally be called an ADP accumulation assay. It detects the displacement of the ADP Tracer, an ADP analog, from the Eu-labeled anti-ADP antibody.  As more ADP is formed in the kinase reaction, more Tracer is displaced. When the Tracer is bound, the TR-FRET signal is high. When the Tracer is displaced, the TR-FRET signal is low. 

The sensitivity of the assay, that is, the amount of ADP that must be formed to displace half of the antibody-bound ADP Tracer (and determine the IC50), depends strongly on the percentage of the antibody that is bound with Tracer under the initial conditions when no ADP is present. 

Compare two cases. Case 1: Initially, 95% of the antibody is bound with Tracer. Case 2: Initially, 50% of the antibody is bound with Tracer. In the first case, significantly more ADP must be formed in order to fully displace Tracer from the antibody. For the second case, it will take less ADP formation to fully displace the Tracer. For case 1, the change in signal or assay window will be larger than in case 2. The IC50 is determined at the point when half of the Tracer is displaced. Since more ADP is required in case 1 to displace half of the Tracer, the IC50 will be right-shifted (less sensitive) compared to case 2. Select an assay window with the least amount of Tracer that will yield a Z’ Factor of >0.5. Do not optimize for maximum assay window. 

Please refer to the kit COA for recommended ADP Tracer concentrations and to the Adapta Universal Kinase Assay User Guide for how to optimize the ADP Tracer concentration.

If you need to compare the potency of compounds to each other, the emission ratios can be used directly to establish rank order potency. If you need to compare the potency of compounds across kinases or with different types of assays, for example the LanthaScreen Activity or LanthaScreen Binding Assays, then the emission ratio should be converted to an IC50.

An IC50 value may be estimated by calculating the EC20 value. The EC20 value is the concentration of inhibitor which causes a 20% change in the Emission Ratio compared to the 0% inhibition value using the plot of the Emission Ratio versus inhibitor concentration. This method is substantially less accurate than when calculating the IC50s from the Emission Ratio using a standard curve, combined with curve fitting software, as described in the next question below “In an Adapta assay, how do I calculate IC50s from the Emission Ratio?”.


The response of Adapta assays to ADP formation is not linear. Therefore, the concentration of inhibitor required to cause a 50% decrease in the assay signal, measured as the emission ratio of the 665 nm to 615 nm RFU is NOT equivalent to the IC50 value of the inhibitor. The EC50 will be right-shifted compared to the IC50. The results of an example that will be worked through are summarized below:

graph showing that response of Adapta assays to ADP formation is not linear

Note: Think this curve is backwards?  It is not. 

Low inhibitor concentrationHigh inhibitor concentration
Lots of ADP producedNo ADP produced
Tracer fully displacedNo tracer displaced
Low TR-FRET signalHigh TR-FRET signal

As more inhibitor is present, less ADP is formed and more ADP tracer stays bound to the antibody.  Therefore, as the concentration of inhibitor increase, the signal increases. 

In an assay in which the signal response is linear (or nearly linear) with respect to product formation, such as the LanthaScreen Eu Kinase Binding Assays, Z’-LYTE Assays and others, the concentration of inhibitor that results in a 50% decrease in the assay signal, the EC50, as determined by the emission ratio is equivalent to the IC50 value for that inhibitor.

Since the response of the Adapta Universal Kinase Assay to ADP formation is not linear, the concentration of inhibitor required to cause a 50% decrease in the emission ratio, the EC50, is NOT equivalent to the IC50 value for the inhibitor.

To convert an inhibitor EC50 value to an IC50 value, use an ATP-ADP standard curve to correct for the non-linear response of the Adapta assay. ADP and ATP are provided in the kit. The curve is generated at constant total ATP plus ADP with known amounts of ATP and ADP. 

Note: The substrate should be included in this titration. 

Please refer to the Adapta Universal Kinase Assay User Guide for directions on how to obtain an ATP-ADP titration curve. 

Conceptually, here is what we are doing to get the IC50

1. Plot the emission ratio of the 665 nm/615 nm RFU to the log of the concentration of the compound, log [inhibitor]. The ½ point on the curve as determined by the software of your choice is the assay EC50.

a. The emission ratio with no inhibitor present is about 0.27.

b. The emission ratio at the EC50 is about 0.56. 

c. The EC50 is 35 nM.

graph showing the use of ATP-ADP standard curve to correct for the non-linear response of the Adapta assay

2. Use the ATP-ADP titration curve to convert the emission ratios to percent of ATP converted.

a. The percent conversion with no inhibitor present at an emission ratio of 0.27, black dashed line below, is 10% conversion of ATP to ADP.

b. The percent conversion at the EC50 with an emission ratio of 0.56, red dashed line, is about 3% conversion of ATP to ADP. This drop from 10% conversion to 3% conversion means that only 30% of the kinase activity is left, or about 70% inhibition. We are looking for 50% inhibition.

Note: If this seems confusing just think of it in arbitrary units. If the kinase activity dropped from 10 to 3, it is clear that the activity has gone down 70%. The confusion comes from different usages of the word percent.

c. A 50% decrease in the activity of the kinase would represent a drop in the ATP conversion from 10% to 5%.  

Note: Really low rates of ATP conversion are normal for this assay and desirable.

graph showing the use of ATP-ADP titration curve to convert the emission ratios to percent of ATP converted

3. Use the ATP-ADP curve again, this time to find 5% conversion of ATP to ADP. 

a. 5% conversion corresponds to an emission ratio of 0.4.  

graph showing use of ATP-ADP curve to find 5% conversion of ATP to ADP

4. Return to the plot of the emission ratio vs. log [inhibitor].

a. An emission ratio of 0.4 yields an IC50 of 12 nM.

Note: As discussed above, the EC50, 35 nM is right-shifted compared to the true value of the IC50, 12 nM.

graph showing the return to the plot of the emission ratio vs log
Assay and Target Preparation

On the product page for your kinase or nuclear receptor, please refer to the Certificate of Analysis for information related to storage of the product.

In general:

  • Do not make single-use aliquots. Activity of the kinase or nuclear receptor can be significantly diminished, probably due to increased surface area of the plastic relative to the volume of the sample.
  • Do not store or refreeze diluted samples as they will quickly lose activity.
  • Do not vortex or shake the kinase or nuclear receptor as it will be denatured.
  • Spinning a sample down into the tube will not generally harm the kinase or nuclear receptor and is helpful to remove material trapped in the cap.

1X refers to the final concentration of any material in the assay under running conditions.

2X, 4X, and other designations refer to reagents that are prepared at higher concentrations before addition to the assay because they will be diluted with other reagents once delivered to the assay plate.

  • A reagent prepared at 2X will be diluted with an equal volume of other reagents in the assay plate.
  • A reagent prepared at 4X will be diluted with three times its volume with other assay components.

Typical example from Z’-LYTE  assays:

  • 2.5 µL of 4X test compound
  • 5 µL of 2X enzyme/substrate
  • 2.5 µL of 4X start reagent such at ATP

We recommend making a 100X serial dilution of the test compound in 100% DMSO solution. Here is the protocol:

1. Begin with a 1 mM stock solution of the test compound in 100% DMSO.

  •     This is the 100X in 100% DMSO starting point.
  •     This is appropriate for the highest 1X starting concentration of 10 µM.

2. In a DMSO-tolerant assay plate, place 20 µL of 100% DMSO in 9 wells, A2–A10, skipping well A1.

3. In well A1, place 30 µL of the 1 mM stock solution.

4. Transfer 10 µL of material from well A1 to A2, mix by pipette.

5. Repeat until there are 10 concentrations, discard the last 10 µL.

Prepare an intermediate dilution of the test compound in assay buffer. This is typically done at 2X, 3X or 4X. Please refer to your specific assay protocol

  • Compounds are typically soluble in 100% DMSO and so as you prepare the serial dilution, the compound will not precipitate before you carry it to the next well.
  • This significantly stabilizes IC50 values from day to day.

This is not possible with the standard assays we offer. However, we do offer custom assay development services by means of which you may be able to get a custom assay format that can be performed in cell lysates, where the cells are engineered to contain a fluorescently tagged downstream target.

Possibly, an IP pull down may be able to be used if it is sufficiently concentrated. Purchaseof the recombinant kinase or nuclear receptor provided by Thermo Fisher Scientific is recommended as a control and to establish that the Thermo Fisher Scientific assay is functioning as expected.

The concentration is lot-specific and is listed in the Certificate of Analysis (COA).

For Research Use Only. Not for use in diagnostic procedures.