UA-Glo® Max Luminescent Kinase Assay

The UA-Glo Kinase Luminescent Detection Kit can quantitatively determine kinase activity in enzymology experiments. This kit measures kinase activity by quantifying the amount of remaining ATP after the kinase reaction: the amount of remaining ATP is negatively correlated with kinase activity. When using this kit, the maximum ATP concentration in the kinase reaction can be up to 10μM; when using the UA-Glo Kinase Plus Luminescent Detection Kit, the maximum ATP concentration in the kinase reaction can be up to 100μM; and when using the UA-Glo Kinase Max Luminescent Detection Kit, the maximum ATP concentration in the kinase reaction can be up to 500μM. 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 between ATP-competitive and non-competitive inhibitors by adjusting the ATP concentration.

Product composition: Luciferase, luciferin, and buffer are mixed and filled into 10 ml or 100 ml brown bottles. The specifications are as follows:

1. Kinase Reaction
1) Perform the kinase reaction in a 96-well or 384-well white opaque assay plate. It is recommended that the reaction volume is 50μL for 96-well plates and 10μL for 384-well plates. Test compounds with concentration gradients can be added to the kinase reaction.
2) The concentrations of kinase and substrate in the kinase reaction 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.
3) ATP concentration: The UA-Glo Kinase Max Luminescent Detection Kit can be used up to 500μM.
4) The kinase reaction can be carried out in a universal reaction buffer (40mM Tris-HCl (pH 7.5), 0.1 mg/ml BSA, 20mM 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. For high-throughput screening experiments of compounds, it is recommended to optimize the kinase reaction to be carried out at room temperature (22℃-25℃) to help maintain the temperature uniformity of the assay plate during the kinase activity determination.

2. Kinase Activity Determination
1) Take out the UA-Glo Kinase Max Luminescent Detection Reagent and equilibrate to room temperature. Mix gently by shaking. The luciferase reaction in the kinase activity luminescent detection reagent is sensitive to temperature changes, so the reagent and the assay plate need to be equilibrated to room temperature, and the temperature during the test should be kept constant (±1℃).
2) If the kinase reaction is carried out at a non-room temperature, such as 30℃, the assay plate needs to be equilibrated to room temperature.
3) Add 50 µL of kinase luminescent detection reagent to a 50 µL 96-well assay plate, or 10 µL of kinase luminescent detection reagent to a 10 µL 384-well assay plate, shake to mix, and place in the dark for 10 minutes.
4) Read the fluorescent signal. Since the fluorescent signal is very stable, if necessary, the plate can be read within 3 hours after adding the detection reagent.

1) It is not recommended to arbitrarily change the dosage of reaction reagents without strict verification. The volume ratio of the kinase reaction to the UA-Glo kinase luminescent detection reagent should be 1:1.
2) After the first use of the UA-Glo kinase luminescent detection reagent, it should be aliquoted and stored at -20°C to ensure the stability of the reagent. There is no loss of signal intensity after 4 repeated freeze-thaw cycles.
3) This product is for research use only.