Adapta® Universal Kinase Assay and Substrates
| The Adapta® Universal Kinase Assay Kit is a homogenous, fluorescence-based immunoassay for the detection of ADP. Adapta® Assays utilize Transcreener® HTS Assay Platform technology (patent no 7332278) under license from Bellbrook Labs, LLC. Some of the benefits of this assay format include: |
- High Z’-factors at low percent conversion of ATP to ADP
- One assay suitable for all kinases, including difficult to assay targets such as lipid kinases
- Best in class Alexa Fluor® dyes enable a superior red-shifted TR-FRET assay
- Reduced interference from compound autofluorescence or light scattering from precipitated compounds
- Complete validated assay system available including substrate and specialized assay buffer
A growing list of Invitrogen kinases have been validated for use with the Adapta® Universal Kinase Assay Kit and corresponding substrates. Please view our Adapta® Assay Reactivity Table to see if your kinase has been validated with the Adapta® Assay.
Assay principle
The principle of the Adapta® Universal Kinase Assay is shown in Figure 1. The assay itself can be divided into two phases: a kinase reaction phase, and an ADP detection phase. In the kinase reaction phase, all kinase reaction components are added to the well and the reaction is allowed to incubate (typically 60 minutes). After the reaction, a detection solution of Europium-labeled anti-ADP antibody, an Alexa Fluor® 647 labeled ADP tracer, and EDTA (to stop the kinase reaction) is added to the assay well. ADP formed by the kinase reaction (in the absence of an inhibitor) will displace the Alexa Fluor® 647 labeled ADP tracer from the antibody, resulting in a TR-FRET signal decrease. In the presence of an inhibitor, the amount of ADP formed by the kinase reaction is reduced, and the resulting intact antibody-tracer interaction results in a high TR-FRET signal.
Figure 1. Schematic principle of the Adapta® Universal Kinase Assay
Assay sensitivity
The Adapta® Universal Kinase Assay measures kinase activity by detecting ADP formation. Most of the signal change occurs in the first 10-20% conversion of ATP to ADP (Figure 2). This is in sharp contrast to kinase assays that measure ATP depletion, in which 20% conversion of ATP to ADP results in only a 20% signal change. As a result, the Adapta® Universal Kinase Assays produces high Z’-factors at low % conversions and is ideally suited for use with kinases with low activity since less ADP has to be formed to achieve an optimal assay window. | Figure 2—Assay signal as a function of percent conversion of ATP to ADP. A representative ATP-ADP titration curve highlights the large change in assay signal at only 20% conversion of ATP to ADP. This makes the Adapta® assay ideally suited for use with kinases with low activity since less ADP has to be formed to achieve an optimal assay window. |
Substrate availability
In addition to the Adapta® Universal Kinase Assay Kit, we offer a selection of lipid and peptide based substrates for use with the Adapta® Assay. Both the lipid and peptide substrates were designed and optmized with assay performance in mind.
Our lipid substrates were designed for optimal performance in the Adapta® Universal Kinase Assay with our PI3 kinases.
- No re-hydrations, sonication, or extrusion needed, the lipid substrates are ready for immediate use in your kinase assay
- Formulated for optimal performance with PI3 kinases
- Get reliable, reproducible performance each and every time, even after multiple freeze-thaw cycles
Since lipid kinases are often highly sensitive to reaction conditions, such as the type and concentration of detergents and salts, we have optimized buffers for each PI3 kinase in our collection.
A list of substrates and buffers currently available from Invitrogen for use in the Adapta® Universal Kinase Assay, can be found below.
Adapta® Assay reactivity table
The following is a list of kinases which have been validated using the Adapta® Universal Assay Kit. Please check back often as we are continuously validating additional kinases.The table also includes the recommended substrate and buffer to run the assay as outlined in the assay conditions section. 5X Kinase Buffer A is included with the purchase of the Adapta® Assay Kit.
Instrument settings
Most instruments, settings, and filters that work with other europium-based TR-FRET assay systems will perform well with the Adapta® Universal Kinase Assay. As with other TR-FRET systems, the europium donor is excited using a 340-nm excitation filter with a 30-nm bandpass. Energy transfer to the Alexa Fluor® 647 tracer is measured using a filter centered at 665 nm with a 10 nm bandpass. This signal is then referenced (or “ratioed”) to the emission from europium peak, using a 615 nm, 10-nm bandpass filter (See Figure 3). The “emission ratio” is calculated as the 665 nm signal divided by the 615 nm signal.For assistance with determining if your instrument is suitable to read the Adapta® Universal Kinase Assay, or for assistance with instrument setup, please call us at 1 760 603 7200 (select option 3, then enter extension 40266).
Figure 3. Excitation and emission spectra of the Adapta® Universal Kinase Assay
The emission spectrum of the europium donor (in black) overlaps with the excitation spectrum for the Alexa Fluor® 647 ADP tracer (dashed red line). Following energy transfer, the emission from the Alexa Fluor® 647 ADP tracer (solid red line) is detected with a filter centered at 665 nm with a 10 nm bandpass. This signal is then referenced (or ‘ratioed’) to the emission of the europium signal using a filter centered at 615 nm with a 10 nm bandpass.
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