Kinases are a large and diverse group of enzymes that are involved in many cellular metabolic and regulatory processes. During the kinase reaction, substrates are phosphorylated while ATP is converted into ADP. Screening for active kinases or for kinase inhibitors is an important tool for the development of new drugs. To fulfil this need, Promega developed a kinase assay that is based on luminescence and the by-product ADP. The ADP-Glo™ Kinase assay is a universal, homogeneous, high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during the kinase reaction. This assay can be used to screen for any ADP-generating enzyme.
In this application note we will show results of the ADP-Glo Kinase assay obtained with multidetection microplate readers from BMG LABTECH.
The principle of the assay is presented in figure 1.
The assay consists of two steps. After the enzymatic reaction is finished, ATP and ADP are present in the well. The first step is to add the ADP-Glo™ Reagent resulting in the elimination of the remaining ATP. The second step is to convert the remaining ADP into ATP by adding Kinase Detection Reagent. This reagent also contains everything needed to measure the newly generated ATP with the help of a luciferase/luciferin reaction. The luminescence measured after incubation is proportional to the ADP concentration generated during the enzymatic reaction.
Materials & Methods
- White 384-well small volume plates from Greiner
- ADP-Glo™ Kinase Assay from Promega, including ATP and ADP
- BMG LABTECH microplate reader
To mimic an enzymatic reaction, an ADP/ATP standard curve was prepared from the nucleotide stock solutions that were supplied with the kit. The assay allows ADP measurements from 1 mM ADP to 0.01 μM ADP. Four different ADP/ATP standard curves were prepared: 1 mM, 100 μM, 10 μM and 1 μM. As an example, the dilution table for a 1 mM ATP/ADP standard dilution is given below. All other standard curves were diluted accordingly.
Five μL of standard dilution was mixed with 5 μL of ADP-Glo™ reagent followed by a 40 min incubation at room temperature. After that 10 μL of Kinase Detection Reagent was then injected with onboard injectors. The signal was measured each minute for 40 minutes. Alternatively it is possible to measure endpoint luminescence after a 40-60 min incubation at room temperature.
Instrument settings for a kinetic reaction
|Detection Mode.||Luminescence, plate mode|
|Measurement time:||1 sec|
|Cycle Time:||60 sec|
|Optics:||dedicated luminescence optic|
|Gain:||Optimized for the ADP concentration|
Results & Discussion
Fig. 2 shows a signal curve for a 1 mM ADP/ATP standard dilution.
The signal curves show that already after 20 min the signal is very stable. The 12 standards show different signal heights according to their ADP concentration. The last 10 data points of the signal curves were averaged and taken as a base for a linear regression fit
Such a fit for a 1 mM nucleotide standard curve can be seen in Fig. 3. The same gain setting was used to create data for a 1 μM standard curve (Fig. 4).
It is possible to measure standard curves from 1 mM to 1 μM ADP using the same gain settings indicating a high assay window.
The data in this application note proves that BMG LABTECH microplate readers can successfully perform the ADP-GloTM Kinase Assay from Promega in 384-well format using only 20 μl. Equipped with on board injectors, it is possible to choose to follow the reaction online using the kinetic mode or as an endpoint measurement.