UniOne® TR-FRET KinActive-BTK Kit
UniOne® TR-FRET KinActive-BTK Kit
Product Details
Product Details
Product Specification
| Host | Human |
| Stability & Storage | -80℃ |
Background
TR-FRET KinActive-BTK Kit is a method that uses time-resolved fluorescence resonance energy transfer (TR-FRET) technology to measureBruton's tyrosine kinase (BTK kinase)activity, suitable for detecting BTK kinase activity and screening inhibitors; the method is easy to perform.
As shown in the figure below, the assay consists of two steps: enzymatic reaction and detection. During the enzymatic reaction, the reaction mixture includes the kinase, biotinylated substrate, and ATP. After adding ATP, the kinase phosphorylates the biotinylated BTK 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.
Components
[Product Components and Storage Conditions]
Reagent Name |
Concentration |
100 Tests |
500 Tests |
2,500 Tests |
10,000 Tests |
BTK Substrate |
20× |
10μL |
50μL |
250μL |
1mL |
BTK |
4.96μM |
5μL |
25μL |
125μL |
500μL |
SA-BAc |
25× |
20μL |
100μL |
500μL |
2mL |
BTK-Antibody-Eu |
25× |
20μL |
100μL |
500μL |
2mL |
supplement component |
400nM |
20μL |
100μL |
500μL |
2mL |
5×Enzymatic Buffer |
5× |
0.4mL |
2mL |
10mL |
40mL |
1×Detection Buffer |
1× |
2mL |
10mL |
50mL |
200 mL |
Note: The product mainly includes biotinylated substrate, detection reagents, and buffers. The buffer can be stored at 4°C, while other components should be stored at -80°C.
Add ingredients (not provided)
The kit components do not include enzyme buffer; use ddH₂O.2O, dilute to 1×, then add DTT, MgCl₂.2, MnCl₂.2Prepare kinase buffer; recommended concentrations are 1 mM DTT, 5 mM MgCl₂.2, 5mM MnCl₂2. The kit does not include ATP; the optimal concentration of ATP must be determined in advance through gradient dilution.
Protocol
[Experimental Procedure and Operations]
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 20μL (the reagent volumes for the reaction system are shown in the table). Calculate the required volume before preparation and prepare only as needed; the following preparation is for reference only, using 500 reactions as an example.
During the enzymatic reaction step, various reagents are diluted with the prepared 1×Enzymatic BufferFor detection reagents, use the provided1×Detection BufferConfiguration. It is not recommended to store working solutions of various components after preparation; they must be used immediately. To prevent degradation, kinase working solutions should be kept on ice during the experiment.
Components |
Preparation Method |
1×Enzymatic Buffer |
Dilute with ddH2O5×Enzymatic BufferDilute to 1×,AddAddsupplement component (final concentration 4 nM), DTT, MgCl2, MnCl2Prepare 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). |
BTK kinase |
Dilute with 1×Enzymatic Bufferto prepare a working solution with a concentration of 5 μM. |
BTK Substrate |
Dilute the BTK Substrate stock solution with 1×Enzymatic Bufferto prepare5×BTK Substrateworking solution,the concentration of this working solution is five times the final concentration of BTK substrate required for the enzymatic reaction step. |
ATP |
Dilute the ATP stock solution with 1× Enzymatic Buffer to preparea 5× ATP working solution; the concentration of this working solution is five times the final ATP concentration required for the enzymatic reaction step. (The ATP concentration must be pre-diluted in gradients to determine the optimal usage concentration.) |
SA-BAc |
Use1×Detection BufferDilute according to the dilution factor until it becomes 1×, which is the working solution. |
BTK-Antibody-Eu |
Dilute with 1× Detection Buffer according to the dilution factor until it becomes 1×, which is the working solution. |
1.2Sample to be tested(Inhibitor) gradient dilution
Dilute the compound using 1× Enzymatic Buffer, and determine the dilution factor and gradient based on the consistent effect of the inhibitor.
2. Reaction system
As shown in the figure, taking a 384-well microplate 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 before reading 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 |
||||
Components |
Experimental well |
Negative control |
Eu-labeled antibody |
Buffer solution |
|
Inhibitor/kinase buffer |
4 μL |
4 μL1×Enzymatic Buffer |
10 μL |
10 μL |
1. Enzymatic reaction (10μL) incubate at room temperature or 37℃ for 30 minutes. |
BTK |
2 μL |
2 μL |
|||
BTK Substrate |
2 μL |
2 μL1×Enzymatic Buffer |
|||
ATP |
2 μL |
2 μL |
|||
SA-BAc |
5 μL |
5 μL |
5 μL 1×Detection Buffer |
10 μL |
2. Detection steps (10μL) Incubate at room temperature for 1 hour. |
Eu-labeled antibody |
5 μL |
5 μL |
5 μL |
||
[Detection]
Detect on a compatible TR-FRET microplate reader (excitation light at 320 nm, detecting emission wavelengths at 620 nm and 665 nm).
[Result Calculation]
1) Calculate the signal value (Ratio): multiply the 665 nm fluorescence signal by the 620 nm fluorescence signal, then multiply by 10,000.
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%
[ResultsDisplay]
1) Kinase activity test
Note: RecommendedMicroplate (384-well plate, white, shallow wells)
