Carna provides ADP-Glo™ assay conditions used to determine compound activity of reference compounds using our kinases. This information is shared to facilitate setting up drug evaluation or screening assays using the ADP-Glo™ Kinase Assay, in your laboratories.

The data obtained under the assay conditions introduced on this page can be viewed in the PDF attached in the Sample Activity Data column. Details such as ATP concentration, metal ion conditions, additives, and reaction time for each target are available in the respective attached PDFs.

• Our ADP-Glo™ Assay Protocol
• The importance of ATPase activity assessment

All the kinase proteins, biotin-labeled peptide substrates, and assay buffer products used to obtain these data are available for purchase from Carna. For product inquiries, questions, or consultations, please contact us from here or at info@carnabio.com.

ADP-Glo™ Kinase Assay Reaction Conditions

Our ADP-Glo™ Assay Protocol

• Prepare the assay buffer (20 mM HEPES, 0.01% Triton X-100, 2 mM DTT, pH 7.5).
• Using the assay buffer, prepare 4x ATP solution, 4x substrate/metal solution containing additives required for each kinase reaction, and 4x kinase solution.
• In each well of a black polystyrene 384-well microplate (Corning #3573), mix 5 µL of assay buffer, 5 µL of 4x ATP solution, 5 µL of 4x substrate/metal solution, and 5 µL of 4x kinase solution. Incubate at room temperature for 1 hour.
• Add 20 µL of ADP-Glo™ reagent (Promega) to each well and incubate for at least 40 minutes to terminate the kinase reaction and decompose residual ATP.
• Add 40 µL of Kinase Detection Reagent (Promega) to each well and incubate for at least 40 minutes to convert ADP to ATP.
• Measure luminescence intensity in each well using EnVision 2104 (Revvity), and evaluate kinase activity by converting luminescence intensity to ADP concentration.

Note: For detailed protocols, please refer to the ADP-Glo™ Kinase Assay Technical Manual provided by Promega.

The importance of ATPase activity assessment

ATPase is an enzyme that catalyzes the hydrolysis of ATP into ADP and inorganic phosphate (Pi) utillizing only water(H2O) as a reactant - without the involvement of any additional molecular components. Carna has observed that in vitro, certain kinases show high intrinsic ATPase activity generating ADP in the absence of peptide substrate. The ADP-Glo™ Kinase Assays convert the ADP produced in the kinase assay back to ATP which is then quantified via a luciferase reaction and measured as a luminescence readout. While the luminescence intensity is utilised as a readout of kinase activity, ADP produced by intrinsic ATPase activity may generate ADP independently of substrate presence. When interpreting ADP-Glo™ data, it is important to bear ATPase activity in mind.

Carna has provided our observed rates of ATPase activity for each of our highly-purified kinases in the table above. These can be used to understand the potential impact of ATPase activity in the ADP-Glo™ assays.

JNK2 (MAPK9) activity was measured using the ADP-Glo™ Kinase Assay with K252a as the substrate.
An increase in RLU was observed in a kinase concentration-dependent manner even in the absence of substrate. Although RLU also increased in the presence of substrate, the majority of the measured luminescence was attributed to ATPase activity, which hydrolyzes ATP, rather than to the specific kinase activity that phosphorylates the substrate. This indicates that the ATPase activity of the kinase is stronger than its substrate-specific phosphorylation activity.

Even when measuring kinase activity using methods other than ADP-Glo™, the use of kinases with strong ATPase activity may lead to the degradation of ATP in the reaction system, potentially affecting the results. Therefore, for such kinases, it is advisable to complement the assay with methods that directly detect substrate phosphorylation, such as the Mobility Shift Assay, to account for the influence of ATP hydrolysis.

JNK2（MAPK9）Mobility Shift Assay Measurement
At an ATP concentration near the Km value (e.g., 50 µmol/L), ATP hydrolysis by ATPase activity may lead to depletion of ATP, resulting in a plateau of the specific activity signal. Under such conditions, substrate phosphorylation becomes undetectable in a kinase concentration-dependent manner. Therefore, for such cases, it is necessary to adjust the enzyme concentration to a level that does not cause signal saturation, enabling accurate measurement of kinase activity.

For those considering the use of ADP-Glo™ assay kits or ADP-Glo™ kinase assay services provided by Carna

Detailed information on ADP-Glo™ products and services offered by Carna is available through the dedicated pages linked below. For any inquiries regarding our products or services, please contact us.

ADP-Glo™ Profiling Assay Services

ADP-Glo™ Assay Kits