UniOne® TR-FRET KinActive-EGFR Kit
UniOne® TR-FRET KinActive-EGFR Kit
Product Details
Product Details
Product Specification
| Host | Human |
| Stability & Storage | -80℃ |
Background
[Background Introduction]
The TR-FRET KinActive-EGFR Kit is a method that uses time-resolved fluorescence resonance energy transfer (TR-FRET) technology to measure the activity of receptor tyrosine kinases,specifically EGFR kinase,and is suitable for detecting EGFR kinase activity and screening inhibitors. This method is easy to operate.
As shown in the figure, the detection method comprises two steps: an enzymatic reaction and a detection step. During the enzymatic reaction, the reaction mixture includes a kinase, a biotinylated substrate, and ATP. Upon addition of ATP, the kinase phosphorylates the biotinylated BTK substrate. In the detection step, the detection reagent is diluted with a detection buffer; the EDTA in the buffer terminates the enzymatic reaction. An 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, and thus to the kinase's phosphorylation activity in the system.
Components
[Product Components and Storage Conditions]
Reagent Name |
Concentration |
100 Tests |
500 Tests |
2,500 Tests |
10,000 Tests |
EGFR Substrate |
20× |
10 μL |
50 μL |
250 μL |
1 mL |
EGFR |
0.3 mg/mL |
5 μL |
25 μL |
125 μL |
500 μL |
SA-BAc |
25× |
20 μL |
100 μL |
500 μL |
2 mL |
EGFR-Antibody-Eu |
25× |
20 μL |
100 μL |
500 μL |
2 mL |
Supplement Component |
400 nM |
20 μL |
100 μL |
500 μL |
2 mL |
5× Enzymatic Buffer |
5× |
0.4 mL |
2 mL |
10 mL |
40 mL |
1× Detection Buffer |
1× |
2 mL |
10 mL |
50 mL |
200mL |
Note: The product mainly includes biotinylated substrate, detection reagents, and buffers. The buffers can be stored at 4°C, while other components should be stored at -80°C.
Additives (not provided)
The kit components do not include enzyme buffer; use ddH₂O.2O, dilute to 1×, then add DTT, MgCl₂.2, MnCl₂.2To prepare the kinase buffer, it is recommended to add 1 mM DTT, 5 mM MgCl₂.22, 5 mM MnCl₂.2. The kit does not contain ATP; the optimal concentration of ATP must be determined in advance through serial dilutions.
Protocol
[Experimental Procedure and Operations]
1. Reagent Preparation
1.1 Before use, allow all reagents to thaw at room temperature (equilibrate at room temperature for at least 30 minutes). The reaction volume in a 384-well plate is 20 µL.muL (the reagent volumes for the reaction system are shown in the table). Calculate the required volume before preparation and prepare accordingly. The following formulation is for reference only, using 500 reactions as an example.
In the enzymatic reaction steps, various reagents are used at a prepared 1× concentration.Enzymatic BufferConfiguration: Use the provided test reagents.1timesDetection BufferPreparation. It is not recommended to store any of the component working solutions after preparation; they must be used immediately. To prevent degradation, kinase working solutions should be kept on ice throughout the experiment.
Component |
Configuration method |
1×Enzymatic Buffer |
Dilute with ddH2O.5timesEnzymatic BufferDilute to 1×.addAddsupplement component(final concentration 2 nM), DTT, MgCl2, Manganese chloride2Prepare as 1× kinase buffer. |
1×Detection Buffer |
1×Detection Buffer |
Inhibitor |
Use 1×Enzymatic BufferDilute the compound to prepare a working solution at 2.5 times the final concentration required for the enzymatic step (10 μL). |
EGFR kinase |
Dilute with 1×Enzymatic Bufferto prepare a working solution with a concentration of 5 μM. |
EGFR Substrate |
Dilute the EGFR Substrate stock solution with 1×Enzymatic Bufferto prepare5×EGFR Substrateworking solution,with a concentration five times the final EGFR Substrate concentration required for the enzymatic reaction step. |
ATP |
Dilute the ATP stock solution with 1× Enzymatic Buffer to prepare5× ATP working solution; this working solution has a concentration five times the final ATP concentration required for the enzymatic reaction step. (The ATP concentration should be pre-diluted in gradients to determine the optimal usage concentration.) |
SA-BAc |
Use1×Detection BufferDilute according to the dilution factor to obtain a 1× working solution. |
BTK-Antibody-Eu |
Dilute with 1× Detection Buffer according to the dilution factor to obtain a 1× working solution. |
1.2Sample to be tested(Inhibitor) gradient dilution
Dilute the compound with 1× Enzymatic Buffer, and determine the dilution factor and dilution 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 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.
Enzymatic reaction |
Control |
|
|||
Component |
Experimental well |
Negative control |
Eu-labeled antibody |
Buffer |
|
Inhibitor/kinase buffer |
4 μL |
4 μL of 1× Enzymatic Buffer |
10 μL |
10 μL |
1. Incubate the enzymatic reaction (10 μL) at room temperature or 37°C for 30 minutes. |
EGFR |
2 μL |
2 μL |
|||
EGFR Substrate |
2 μL |
2 μL of 1× Enzymatic Buffer |
|||
ATP |
2 μL |
2 μL |
|||
SA-BAc |
5 μL |
5 μL |
5 μL 1× Detection Buffer |
10 μL |
2. Detection steps: Incubate at room temperature for 1 hour (10 μL). |
Eu-labeled antibody |
5 μL |
5 μL |
5 μL |
||
[Detection]
Detect on a compatible TR-FRET microplate reader (excitation at 320 nm, emission wavelengths at 620 nm and 665 nm).
[Result Calculation]
1) Calculate the signal value (Ratio): Multiply the 665 nm fluorescence signal by 10000 and divide it by the 620 nm fluorescence signal.
Ratio = (665/620)×10000
2) Calculate based on the signal valueNet signal:
Net signal= (Std-NC)/NC × 100
3) Calculate CV (%):
CV (%) = Standard Deviation / Mean Ratio × 100%
[Result Presentation]
1) Kinase activity test
Note: RecommendedMicroplate (384-well plate, white, shallow wells)
