UA-Glo® Max Luminescent Kinase Assay

The UA-Glo Kinase Luminescence Assay Kit quantitatively measures kinase activity in enzymatic experiments. This kit determines kinase activity by quantifying the remaining ATP after the kinase reaction: the amount of residual ATP is inversely correlated with kinase activity. When using this kit, the ATP concentration in the kinase reaction can be up to 10μM, while the UA-Glo Kinase Plus Luminescence Assay Kit allows ATP concentrations up to 100μM, and the UA-Glo Kinase Max Luminescence Assay Kit supports ATP concentrations up to 500μM. This kit can detect most kinases, with kinase substrates including peptides, proteins, lipids, or sugars. It is suitable for high-throughput homogeneous screening of kinase inhibitors and can differentiate between ATP-competitive and non-competitive inhibitors by adjusting ATP concentrations.

Product Composition: Luciferase, luciferin, and buffer mixture are filled into 10 ml or 100 ml brown bottles. Specifications are as follows:

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1. Kinase Reaction
1) Perform the kinase reaction in a 96-well or 384-well white opaque assay plate. The recommended reaction volume is 50 µL for a 96-well plate and 10 µL for a 384-well plate. Test compounds with concentration gradients can be added to the kinase reaction.
2) The kinase and substrate concentrations for the kinase reaction need to be optimized based on the specific kinase. To achieve the desired signal-to-noise ratio, a kinase concentration within the linear range of the signal can be used.
3) ATP concentration: The UA-Glo Kinase Max Luminescence Detection Kit can accommodate up to 500 µM.
4) The kinase reaction can be performed in a universal reaction buffer (40 mM Tris-HCl (pH 7.5), 0.1 mg/mL BSA, 20 mM MgCl2) or in a reaction buffer and cofactors 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 the kinase reaction at room temperature (22°C–25°C) to maintain uniform plate temperature during kinase activity measurement.

2. Kinase Activity Measurement
1) Take out the UA-Glo Kinase Max Luminescence Detection Reagent and equilibrate it to room temperature. Mix gently by shaking. The luciferase reaction in the kinase activity detection reagent is sensitive to temperature changes, so the reagent and assay plate must be equilibrated to room temperature, and the temperature should remain constant (±1°C) during testing.
2) If the kinase reaction was performed at a non-room temperature, such as 30°C, the assay plate must be equilibrated to room temperature.
3) Add 50 µL of the kinase detection reagent to a 50 µL reaction in a 96-well plate or 10 µL of the kinase detection reagent to a 10 µL reaction in a 384-well plate. Mix by shaking and incubate in the dark for 10 minutes.
4) Measure the luminescence signal. Since the luminescence signal is highly stable, the plate can be read within 3 hours after adding the detection reagent if needed.