UA-Glo® 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 mixed and filled in 10 ml or 100 ml brown bottles. Specifications are as follows:

1. Kinase Reaction
1) Perform the kinase reaction in a 96-well or 384-well white opaque assay plate. A reaction volume of 50 µL is recommended for 96-well plates, and 10 µL for 384-well plates. A concentration gradient of test compounds can be added to the kinase reaction.
2) The concentrations of kinase and substrate in the kinase reaction need to be optimized for different kinases. Under the condition of achieving the required signal-to-noise ratio, experiments can be conducted using kinase concentrations within the linear range of the signal.
3) ATP concentration: The UA-Glo Kinase Luminescence Detection Kit can accommodate up to 10 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 experiments, it is recommended to optimize the kinase reaction at room temperature (22°C–25°C) to ensure uniform temperature across the assay plate during kinase activity measurement.

2. Kinase Activity Assay
1) Take out the UA-Glo Kinase Luminescence Detection Reagent and equilibrate it to room temperature. Gently shake to mix. The luciferase reaction in the kinase activity luminescence 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 (e.g., 30°C), the assay plate should be equilibrated to room temperature.
3) Add 50 µL of the kinase luminescence detection reagent to 50 µL in a 96-well assay plate, or 10 µL of the reagent to 10 µL in a 384-well assay plate. Mix by shaking and incubate in the dark for 10 minutes.
4) Read the luminescence signal. Due to the stability of the fluorescence signal, if necessary, the plate can be read within 3 hours after adding the detection reagent.

1) It is not recommended to arbitrarily alter the reagent quantities without rigorous validation. The volume ratio between the kinase reaction and UA-Glo kinase luminescence detection reagents should be 1:1.

2) After initial use, the UA-Glo kinase luminescence detection reagent should be aliquoted and stored at -20°C to ensure stability. No signal intensity loss is observed after 4 freeze-thaw cycles.

3) This product is intended for research use only.