UA-Glo® Kinase ADP Assay with PI3K Substrate PIP2:3PS

Phosphatidylinositol (4,5)-bisphosphate (PIP2) and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) are derivatives of phosphatidylinositol (PI). PIP2 and PIP3 participate in various cellular physiological processes through dynamic phosphorylation/dephosphorylation. Phosphoinositide 3-kinase (PI3K) is a key enzyme that mediates signaling by phosphorylating phosphatidylinositol derivatives, and its activated PI3K/Akt signaling pathway plays a central role in cell proliferation, metabolic regulation, and immune responses. A class of phosphoinositide 3-kinases (PI3K Class I) generates PIP3 by phosphorylating PIP2, which can participate in downstream cascades mediated by growth factor receptors. Abnormalities in this signaling pathway are highly correlated with mechanisms of tumor development.

The UA-Glo® Kinase ADP Detection Assay quantitatively measures kinase activity in enzymatic assays. This assay determines kinase activity by quantifying the amount of ADP, a product of the kinase reaction: the amount of ADP is directly proportional to kinase activity. The UA-Glo® Kinase ADP Detection Assay can be used with ATP concentrations up to 1 mM in kinase reactions. This kit can detect most kinases, with substrates including peptides, proteins, lipids, or sugars. It is suitable for high-throughput homogeneous screening of kinase inhibitors and can distinguish between ATP-competitive and non-competitive inhibitors by adjusting ATP concentrations. The kit can also be used to measure the activity of any ADP-producing enzyme, such as ATPase.

The UA-Glo® Kinase ADP Detection Kit (with PI3K substrate PIP2) includes the kinase ADP detection reagent and optimized PI3K substrate PIP2:3PS, making it suitable for high-throughput screening of PI3K (Class I) inhibitors.

UA-Glo® KinaseADP Assay Kit (ContainingPI3KSubstratePIP2:3PS) Components and specifications are as follows. Detectable96-/384-well plate reaction count per well25μL / 5μL kinase reaction calculation.

The PI3K kinase reaction is performed in 96-well or 384-well white opaque assay plates. The recommended reaction volume is 25μL for 96-well plates and 5μL for 384-well plates. Test compounds with concentration gradients can be added to the kinase reaction. The PI3K kinase reaction is recommended to be carried out at room temperature (23°C-25°C) for 1 hour, or under customer-optimized temperature and time conditions. No additional reagents are required to terminate the kinase reaction after completion. If termination reagents are needed due to special experimental requirements, avoid using magnesium ion chelators such as EDTA. The UA-Glo® kinase ADP detection reaction requires magnesium ions.

The kit includes a 5x PI3K kinase reaction buffer. Users may also use their own optimized PI3K kinase reaction buffer and cofactors. It is recommended to prepare the PI3K kinase reaction buffer as a 5x stock solution.

The concentration of PI3K kinase in the reaction should be optimized according to the supplier's instructions. For experiments requiring an appropriate signal-to-noise ratio, the kinase concentration within the linear signal response range can be used.

The final concentration of PIP2:3PS substrate in the PI3K kinase reaction is 0.05mg/mL. The recommended ATP concentration is 25μM or a user-optimized concentration. Some commercially available ATP contains residual ADP. Due to the high sensitivity of the UA-Glo® kinase ADP detection reagent, residual ADP in ATP may cause high background. Therefore, high-purity ATP should be used for the kinase reaction, such as the ATP provided in the kit or other high-purity ATP (e.g., Sigma-Aldrich ATP, Cat# A2383, purity ≥99%) or higher-purity ATP.

The following experimental example demonstrates the testing of a PI3K inhibitor IC50, with a PI3K kinase reaction volume of 25μL.

PI3K Kinase Reaction

1)Prepare 10x final concentration of 2x or 3x serially diluted test compounds. Add 2.5μL/well of the diluted test compounds to a 96-well white (opaque bottom) assay plate. Include solvent control wells, positive compound control wells, and no-enzyme control wells. The final concentration of solvents such as DMSO in the kinase reaction should not exceed 1%.

2)The PI3K kinase reaction buffer stock solution is 5x. Dilute it 1:1 with sterile ultrapure water to prepare a 2.5x solution as needed.

3)The PIP2:3PS substrate stock solution is 20x (1mg/mL). Dilute it to 2.5x PIP2:3PS substrate using the 10x PIP2:3PS dilution buffer as follows:

a)Thaw the PIP2:3PS substrate stock solution and equilibrate to room temperature.

b)Vortex vigorously for at least 1 minute to mix.

c)Dilute to 2.5x PIP2:3PS substrate using the 10x PIP2:3PS dilution buffer. For example, add 125μL PIP2:3PS substrate to 250μL 10x PIP2:3PS dilution buffer, then add sterile ultrapure water to 1mL.

d)Vortex vigorously for at least 1 minute to mix.

4)Mix the 2.5x PI3K kinase reaction buffer from step 2 and the 2.5x PIP2:3PS substrate from step 3 in a 1:1 ratio. This is the 1.25x PI3K kinase reaction buffer/PIP2:3PS. Vortex to mix.

5)Add PI3K to the 1.25x PI3K kinase reaction buffer/PIP2:3PS at the optimized enzyme concentration. Pipette or invert to mix. This is the 1.25x PI3K kinase working solution.

6)Add 20μL/well of the 1.25x PI3K kinase working solution to the 96-well plate prepared in step 1 (containing 2.5μL/well of 10x final concentration serially diluted test compounds). For no-enzyme control wells, add 20μL/well of 1.25x PI3K kinase reaction buffer/PIP2:3PS.

7)Incubate at room temperature for 10-20 minutes to allow binding of test compounds to PI3K.

8)Initiate the kinase reaction by adding ATP: Dilute the kit-provided 10mM ATP to 250μM with sterile ultrapure water. Add 2.5μL/well of 250μM ATP to the 96-well plate from step 7. The final ATP concentration is 25μM, and the kinase reaction volume is 25μL.

9)Shake the plate to mix and incubate at room temperature (23°C-25°C) for 1 hour.

10)Proceed to ADP detection as follows.

ADP Detection After PI3K Kinase Reaction

1.ATP Removal After Kinase Reaction

1)Thaw the UA-Glo® kinase ATP removal reagent and equilibrate to room temperature. Invert to mix.

2)Add MgCl2 to the UA-Glo® kinase ATP removal reagent to a final concentration of 10mM. Invert to mix.

3)If the kinase reaction was performed at non-room temperature (e.g., 30°C), equilibrate the plate to room temperature. The luciferase reaction in the ADP detection reagent is sensitive to temperature changes. Reagents and test samples/plates must be equilibrated to room temperature, and the testing process should maintain a constant temperature (±1°C).

4)Add 25 µL/well of ATP removal reagent to the 96-well plate containing 25 µL/well of kinase reaction. Shake to mix. The volume ratio of kinase reaction, ATP removal reagent, and ADP detection reagent should be 1:1:2.

5)Incubate at room temperature for 40 minutes.

2.ADP Detection

1)Thaw the UA-Glo® kinase ADP detection reagent and equilibrate to room temperature. Invert to mix. The ADP detection reagent may have slight precipitation after thawing. The supernatant can be used directly or centrifuged to remove precipitation.

2)Add 50 µL/well of ADP detection reagent to the 96-well plate after ATP removal. Shake to mix. Incubate in the dark at room temperature for 40 minutes.

3)Measure the luminescence signal using a luminometer or multifunctional plate reader. The signal can be read as early as 30 minutes after adding the ADP detection reagent. Due to the stable signal, the plate can be read within 3 hours or longer if needed.

1) Different batches are not recommended for mixed use 2) Without strict validation, it is not recommended to change the dosage of detection reagents 3) For research use only