Product Details
Product Details
Product Specification
| Stability & Storage |
Dry ice transportation. Store in the dark at -20℃ or below, with a validity period of 12 months. |
Background
The UA-Glo Kinase Luminescent Detection Kit can quantitatively determine the activity of kinases in enzymology experiments. This kit measures the kinase activity by quantitatively detecting the amount of remaining ATP after the kinase reaction: the amount of remaining ATP is negatively correlated with the 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.
Components
Product composition: Luciferase, luciferin, and buffer are mixed and filled into 10 ml or 100 ml brown bottles. The specifications are as follows:
|
96-well plate reaction count |
384-well plate reaction count |
|
|
10 ml |
200 |
1,000 |
|
100 ml |
2,000 |
10,000 |
|
10 x 100 ml |
20,000 |
100,000 |
Protocol
1. Kinase reaction
1) Perform kinase reactions in 96-well or 384-well white opaque assay plates. It is recommended that the reaction volume for 96-well plates be 50μL and that for 384-well plates be 10μL. Test compounds with concentration gradients can be added to the kinase reactions.
2) The concentrations of kinases and substrates 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 response range of the signal can be used for the experiment.
3) Concentration of ATP: The maximum concentration detectable by the UA-Glo Kinase Plus Luminescent Assay Kit can reach 100μM.
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 compound high-throughput screening experiments, 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 process.
2. Kinase activity assay
1) Take out the UA-Glo Kinase Plus Luminescent Assay Reagent and equilibrate it to room temperature. Shake gently to mix. The luciferase reaction in the kinase activity luminescent assay reagent is sensitive to temperature changes; the reagent and the assay plate need to be equilibrated to room temperature, and the temperature should be kept constant (±1°C) during the test.
2) If the kinase reaction is carried out at a non-room temperature, such as 30°C, 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 well, 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.
Guidelines
1) It is not recommended to arbitrarily change the amount 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 using the UA-Glo Kinase Luminescent Detection Reagent for the first time, 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.
