UA-Glo® Kinase ADP Assay

The UA-Glo Kinase ADP Detection Kit enables quantitative measurement of kinase activity in enzymatic assays. This kit determines kinase activity by quantitatively detecting the amount of ADP produced in the kinase reaction: the level of ADP is directly proportional to kinase activity. The kit supports ATP concentrations up to 1 mM in the kinase reaction. It is compatible with a wide range of kinases, using substrates such as peptides, proteins, lipids, or sugars.

The kit is suitable for homogeneous high-throughput screening of kinase inhibitors and can distinguish between ATP-competitive and non-competitive inhibitors by adjusting ATP concentrations. It can also be used to measure the activity of any ADP-generating enzyme, such as ATPase.

Key features of the kit include high signal-to-noise ratio, excellent reproducibility, and superior stability. Its homogeneous, "add-and-read" format reduces preparation and handling steps, minimizing errors associated with multiple pipetting steps. The stable glow-type luminescence signal makes it particularly suitable for high-throughput compound screening.

The kit works across a broad ATP concentration range (1 µM to 1 mM) and can differentiate between competitive and non-competitive kinase inhibitors. With high sensitivity and a wide dynamic range, it can detect as little as 0.25 pmol of ADP. This allows for lower kinase consumption in assays and facilitates optimization of HTS conditions.

The simplified procedure involves a homogeneous detection step after the kinase reaction, making the workflow straightforward and easy to perform—ideal for high-throughput screening applications. The fluorescence signal remains stable and linear for 0.5 to 12 hours, supporting large-scale multi-well plate experiments. In addition, the proprietary formulation minimizes interference from test compounds.

1. Kinase Reaction

1)  Perform the kinase reaction in a white, opaque 96-well or 384-well plate. A reaction volume of 25 µL for 96-well plates and 5 µL for 384-well plates is recommended. A concentration gradient of the test compound may be included in the kinase reaction.

2)  The concentrations of the kinase and its substrate should be optimized for each specific kinase. To achieve an adequate signal-to-noise ratio, the kinase concentration should be within the linear range of the signal response. Due to the high sensitivity of the UA-Glo Kinase ADP Detection Kit, the amount of kinase required can be significantly reduced.

3)  Important Note: The ATP concentration can be used at up to 1 mM. Some commercially available ATP sources contain ADP contaminants. Given the high sensitivity of the UA-Glo Kinase ADP Detection Kit, residual ADP can lead to a high background signal. Therefore, it is critical to use high-purity ATP, such as Sigma-Aldrich Cat# A2383 (purity ≥99%) or other ATP of equivalent or higher purity.

4)  The kinase reaction can be performed in a universal reaction buffer (e.g., 40 mM Tris-HCl (pH 7.5), 0.1 mg/ml BSA, 20 mM MgCl₂) or in a buffer system with cofactors as reported in the literature.

5)  The temperature and duration of the kinase reaction should be set according to the specific kinase. For high-throughput compound screening, it is recommended to optimize and perform the kinase reaction at room temperature (22°C–25°C) to maintain consistent temperature across the plate during the subsequent ADP detection step.

6)  No additional reagent is required to terminate the kinase reaction after its completion. If a termination step is necessary for specific experimental requirements, avoid using magnesium chelators (e.g., EDTA), as magnesium ions are essential for the subsequent UA-Glo ADP detection reaction.

2. ATP Removal Post-Kinase Reaction

1)  Take out the ATP Removal Reagent and equilibrate it to room temperature (22°C–25°C). Mix gently by swirling.

2)  If the kinase reaction was performed at a non-room temperature (e.g., 30°C), equilibrate the reaction plate to room temperature.

3)  Add 25 µL of the ATP Removal Reagent to the 25 µL kinase reaction in the 96-well plate (total volume 50 µL), or add 5 µL of the ATP Removal Reagent to the 5 µL kinase reaction in the 384-well plate (total volume 10 µL). Mix thoroughly by shaking.

4)  Incubate at room temperature for 40 minutes.

3. Kinase Activity Measurement (ADP Detection)

1)  Take out the ADP Detection Reagent and equilibrate it to room temperature. Mix gently by swirling.

    Note: The luciferase reaction in the ADP Detection Reagent is sensitive to temperature changes. Both the reagent and the assay plate must be equilibrated to room temperature (22°C–25°C), and the temperature should remain constant (±1°C) during the measurement.

2)  Add 50 µL of the ADP Detection Reagent to the 50 µL reaction mixture in the 96-well plate, or add 10 µL of the ADP Detection Reagent to the 10 µL reaction mixture in the 384-well plate. Mix thoroughly by shaking. Incubate the plate in the dark at room temperature for 30 minutes.

3)  The luminescence signal can be read between 30 and 60 minutes after adding the ADP Detection Reagent. Due to the highly stable luminescent signal, the plate can be read up to 3 hours after reagent addition if necessary.