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View additional product information for Adapta™ Universal Kinase Assay Kit - FAQs (PV5099)
9 product FAQs found
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.
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 (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/adapta_userguide_man.pdf) for directions on how to obtain an ATP-ADP titration curve.
Conceptually, here is what we are doing to get the IC₅₀.
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.
- The emission ratio with no inhibitor present is about 0.27.
- The emission ratio at the EC₅₀ is about 0.56.
- The EC₅₀ is 35 nM.
Use the ATP-ADP titration curve to convert the emission ratios to percent of ATP converted.
- 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.
- 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.
- 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.
Use the ATP-ADP curve again, this time to find 5% conversion of ATP to ADP.
- 5% conversion corresponds to an emission ratio of 0.4.
Return to the plot of the emission ratio vs. log [inhibitor].
- 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.
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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 and combined with curve fitting software.
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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.
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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 (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/adapta_userguide_man.pdf) for how to optimize the ADP Tracer concentration.
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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.
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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
(https://assets.thermofisher.com/TFS-Assets/LSG/manuals/adapta_userguide_man.pdf).
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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.
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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, linked below:
https://www.thermofisher.com/us/en/home/industrial/pharma-biopharma/drug-discovery-development/target-and-lead-identification-and-validation/kinasebiology/kinase-activity-assays/adapta-universal-kinase-assay.html#reactivity
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The Adapta Universal Kinase Assay Kit (https://www.thermofisher.com/order/catalog/product/PV5099) 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.
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