UniOne® TR-FRET KinActive-TK general Kit

UniOne® TR-FRET THUNDER™ KinActive-TK general Kitis a universal method that uses time-resolved fluorescence resonance energy transfer (TR-FRET) technology to measure tyrosine kinase activity, suitable for detecting the activity of various tyrosine kinases and screening inhibitors. This method is easy to operate.

 As shown in the figure below, the assay consists of two steps: enzymatic reaction and detection. During the enzymatic reaction, the reaction mixture includes kinase, biotinylated substrate, and ATP. After adding ATP, the kinase phosphorylates the biotinylated TK substrate. In the detection step, the detection reagents are diluted with the detection buffer; the EDTA in the buffer terminates the enzymatic reaction. The Eu-labeled antibody and streptavidin-BAc simultaneously capture the phosphorylated substrate. The TR-FRET signal generated by the two detection reagents bound to the substrate is proportional to the level of substrate phosphorylation, which in turn is proportional to the kinase's phosphorylation activity in the system.

[Product Composition and Storage Conditions]

Note: The product mainly includes biotinylated substrates, detection reagents, and buffers. Buffers can be stored at 4°C, while other components should be stored at -80°C.

Add components (not provided)

Kit components not includedEGFRKinase, enzyme buffer with ddH₂O₂After diluting to 1×, add DTT, MgCl₂₂, MnCl₂₂Prepare the kinase buffer; recommended concentrations are 1 mM DTT, 5 mM MgCl₂₂, 5 mM MnCl₂₂. The kit does not include ATP; the optimal ATP concentration must be determined in advance through serial dilutions.

1. Reagent Preparation

1.1 Before use, thaw all reagents at room temperature (allow them to equilibrate at room temperature for at least 30 minutes). The reaction system in a 384-well plate is 20 μL (the reagent volumes for the reaction system are shown in the table below). Calculate the required volume for your experiment before preparing and prepare only the amount needed; the following preparation instructions are for reference only, using 500 reactions as an example.

During the enzymatic reaction step, all reagents should be prepared using the configured 1× Enzymatic Buffer, and the detection reagents should be prepared using the provided 1× Detection Buffer. It is not recommended to store working solutions of any components after preparation; they must be used immediately. To prevent degradation, kinase working solutions should be kept on ice throughout the experiment.

1.2 Gradient Dilution of Samples to Be Tested (Inhibitors)

Dilute the compounds using 1× Enzymatic Buffer, and determine the dilution factor and gradient based on the consistent inhibitory effect of the inhibitor.

2. Reaction System

As shown in the figure, taking a 384-well plate as an example, add the inhibitor, substrate, kinase, and ATP, then incubate at room temperature or 37°C (the incubation time and temperature for the enzymatic reaction should be optimized according to the target kinase) for 30 minutes (in the enzymatic reaction system, it is recommended that the DMSO concentration does not exceed 2%). After the reaction, add the detection reagent; the EDTA in the detection buffer will terminate the enzymatic reaction. Incubate at room temperature for 1 hour, then read the values on the instrument.

The experiment should include the following control wells:

 (1) Negative control: non-specific signal, used to calculate the specific signal value of the reaction system.

 (2) Buffer control: to eliminate the influence of the buffer on the signal value.

(3)Eu-labeled antibody control: used to verify the normal signal value of the Eu-labeled antibody at 620 nm.

[Detection]

Detect on a compatible TR-FRET microplate reader (excitation at 320 nm, emission wavelengths at 620 nm and 665 nm).

[Result Calculation]

1) Ratio:

Ratio = (665/620) × 10000

2) Net signal:

Net signal = (Std-NC)/NC × 100

Std: the ratio of sample to control

NC: the ratio of negative control

3) CV (%):

CV (%) = Standard Deviation / Mean Ratio × 100%

Note: Microplates are recommended (384-well plates, white, shallow wells)

[Kinase Assay Optimization]

1)Optimization of supplement component concentration

Some tyrosine kinases may require the addition of a supplement component to the 1X kinase buffersupplement componentreagent to achieve optimal enzyme activity. This step can determine the optimalsupplement componentConcentration—refers to the concentration corresponding to when the signal reaches its maximum value of 80%. Prepare a series of solutions with different concentrations.supplement componentof 1X kinase buffer (recommended range from 125 nM to 0 nM).

2)Kinase concentration optimization

This step is primarily aimed at determining the optimal enzyme concentration (at which the signal reaches 80% of its maximum). A balance should be struck between high detection signal and enzyme consumption. Use a fixed concentration of TK-substrate-biotin (e.g., 1 µM) and ATP (e.g., 100 µM), perform serial dilutions of the enzyme, and test them (recommended final enzyme concentrations are 30, 10, 0.3, and 0 nM; these can also be adjusted flexibly based on preliminary data). Allow the enzymatic reaction to proceed for 30 minutes. The recommended biotin/streptavidin ratio is 8:1.

3)Kinetic studies

The kinetic properties of the enzyme depend on the concentrations of the kinase and the substrate. In incubation time studies, a fixed concentration of kinase (determined from previous experiments), ATP (100 μM), and substrate (1 µM) should be used; the reaction is terminated at different time points by adding the detection reagent at 1, 2, 5, 10, 30, and 60 minutes. The recommended ratio of biotin to streptavidin is 8:1.

4)Substrate concentration optimization

Use the optimal enzyme concentration and a saturated ATP concentration (100 µM). It is recommended to test different concentrations of the TK substrate-biotin, with a concentration gradient ranging from 2 µM to 1 nM (two-fold serial dilutions). The kinase reaction should be terminated at the previously determined optimal incubation time. During the detection step, adjust the streptavidin concentration according to the concentration of the TK substrate-biotin to ensure that the biotin/streptavidin ratio remains at 8:1. Since background signal may increase with higher streptavidin concentrations, a negative control must be set up for each streptavidin sample.

5) ATP concentration optimization

This step optimizes the ATP concentration, requiring the use of the optimal enzyme concentration and a saturated TK substrate concentration (1 µM). It is recommended to test ATP concentrations ranging from 300 µM to 1.7 nM (in threefold serial dilutions). At the optimal incubation time, terminate the kinase reaction by adding the detection reagent. The recommended ratio of biotin to streptavidin is 8:1.

6)Biotin/Streptavidin Ratio Optimization

Optimizing the biotin/streptavidin ratio is a critical step that can significantly enhance signal intensity. Prepare streptavidin solutions with different biotin/streptavidin ratios (2/1, 4/1, 8/1); in the experiment, use the optimal enzyme concentration, ATP concentration, and substrate concentration for detection. Additionally, each streptavidin concentration should include a corresponding negative control, as this reagent directly affects the background value.

7) IC50 determination

Kinase activity must be tested across a broad range of inhibitor concentrations to generate a dose-response curve; this assay is typically performed under previously established optimal assay conditions.