UA-Glo® Kinase ADP Assay

The UA-Glo Kinase ADP Detection Kit can quantitatively determine the activity of kinases in enzymatic experiments. This kit measures the kinase activity by quantitatively detecting the amount of ADP, a product of the kinase reaction: the amount of ADP is positively correlated with the kinase activity. The concentration of ATP in the kinase reaction can be as high as 1 mM. This kit can detect most kinases, and kinase substrates include polypeptides, proteins, lipids, or sugars, etc. The kit can be used for high-throughput homogeneous screening of kinase inhibitors, and can distinguish ATP-competitive and non-competitive inhibitors by adjusting the ATP concentration. It can also be used to detect the activity of any enzyme that produces ADP, such as ATPase. This reagent has the characteristics of high signal-to-noise ratio, good repeatability, and good stability. The homogeneous ready-to-use formula reduces experimental preparation and operation steps, lowers errors caused by multiple sample additions, and its stable glow signal makes this product particularly suitable for high-throughput compound screening.

ATP concentrations ranging from 1 μM to 1 mM can be used in experiments, and can distinguish between competitive and non-competitive kinase inhibitors.

Extremely high sensitivity and a larger dynamic range
ADP as low as 0.25 pmol can be detected. The extremely high sensitivity and larger dynamic range allow for a smaller amount of kinase to meet experimental needs and facilitate the optimization of HTS experimental conditions.

Convenient experimental procedures
The homogeneous detection after the kinase reaction makes the entire experimental process simpler and easier to operate, which is beneficial for carrying out HTS screening experiments.

Suitable for high-throughput detection
Fluorescent signals can be detected within 0.5-12 hours, and the fluorescence intensity maintains a stable linear relationship, meeting the needs of detecting a large number of experimental plates at one time. The proprietary reagent formulation makes this luminescent detection less susceptible to compound interference.

1. Kinase Reaction

1) Kinase reactions are performed in 96-well or 384-well white opaque assay plates. It is recommended that the reaction volume for 96-well plates is 25μL and for 384-well plates is 5μL. Test compounds with concentration gradients can be added to the kinase reaction.

2) The concentrations of kinase and substrate in kinase reactions need to be optimized according to different kinases. Under the condition of the appropriate signal-to-noise ratio required for the experiment, the kinase concentration within the linear reaction range of the signal can be used for the experiment. Due to the high sensitivity of the UA-Glo ADP Kinase Detection Kit, the amount of kinase used can be significantly reduced.

3) Important note: The concentration of ATP can be as high as 1 mM. Some commercially available ATP contains residual ADP. Due to the high sensitivity of the UA-Glo ADP Kinase Assay Kit, residual ADP in ATP will cause a high experimental background. Therefore, high-purity ATP must be used for the kinase reaction, such as ATP sold by Sigma-Aldrich,  Cat# A2383 (purity ≥ 99%) or other ATP with higher purity.

4) Kinase reactions can be performed in a universal reaction buffer (40 mM Tris-HCl (pH 7.5), 0.1 mg/ml BSA, 20 mM MgCl₂) or using reaction buffers and cofactors reported in the literature.

5) The temperature and time of the kinase reaction need to be set according to different kinases. When conducting high-throughput compound screening experiments, it is recommended to optimize the kinase reaction to be carried out at room temperature (22℃-25℃), which is beneficial for maintaining the temperature uniformity of the assay plate during the kinase ADP detection process.

6) No additional reagents are required to terminate the kinase reaction after it ends. If it is necessary to add a kinase reaction termination reagent due to special experimental requirements, avoid using magnesium ion chelators, such as EDTA. Magnesium ions are required for the reaction in the UA-Glo Kinase ADP Detection.

2. Removal of ATP after kinase reaction

1) Take out the kinase ATP removal reagent, equilibrate to room temperature (22℃-25℃). Shake gently to mix well.

2) If the kinase reaction is carried out at a non-room temperature, such as 30°C, equilibrate the assay plate to room temperature.

3) Add 25 µL of ATP removal reagent to the above 25 µL 96-well assay plate (total volume 50 µL), or 5 µL of ATP removal reagent to the 5 µL 384-well assay plate (total volume 10 µL). Mix by shaking. 

4) Incubate at room temperature for 40 minutes.

3. Kinase Activity Assay (ADP Detection)

1) Take out the kinase ADP detection reagent and equilibrate it to room temperature. Shake gently to mix well. (Note: The luciferase reaction in the ADP detection reagent is sensitive to temperature changes. The reagent and the assay plate need to be equilibrated to room temperature (22℃-25℃), and the temperature should be kept constant (±1℃) during the test.)

2)Add 50 µL of ADP detection reagent to the above 50 µL 96-well assay plate, or 10 µL of ADP detection reagent to the above 10 µL 384-well assay plate, and mix by shaking. Incubate at room temperature in the dark for 30 minutes.

3) The fluorescent signal can be read 30-60 minutes after adding the ADP detection reagent. Since the fluorescent signal is very stable, if necessary, the plate can be read within 3 hours after adding the detection reagent.