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Description
UniOne® TR-FRET Human TSLP/TSLPR Binding KitProduct Specification Host Human Stability & Storage 80 Background The kit employs homogeneous time resolved fluorescence (TR FRET) technology to measure the interaction between TSLP and TSLPR. This method enables simple, rapid, and high throughput screening of inhibitors and antibody blockers. As shown in the figure, the interaction between TSLP and TSLPR is detected using Eu labeled anti Tag1 antibody (TR FRET donor) and Ac labeled anti Tag2
Product Specification
| Host | Human |
| Stability & Storage | -80℃ |
Background
The kit employs homogeneous time-resolved fluorescence (TR-FRET) technology to measure the interaction between TSLP and TSLPR. This method enables simple, rapid, and high-throughput screening of inhibitors and antibody blockers.
As shown in the figure, the interaction between TSLP and TSLPR is detected using Eu-labeled anti-Tag1 antibody (TR-FRET donor) and Ac-labeled anti-Tag2 antibody (TR-FRET acceptor). The binding of TSLP and TSLPR brings the donor and acceptor antibodies into proximity, allowing the excitation of the donor antibody to trigger fluorescence resonance energy transfer (FRET) to the acceptor antibody, resulting in a specific emission signal at 665 nm. The positive control drug Bosakitug blocks the binding of TSLP and TSLPR, preventing FRET signal generation. The stronger the blocking effect of the screened drug on TSLP-TSLPR interaction, the lower the signal. This specific signal is proportional to the degree of TSLP-TSLPR interaction. The homogeneous assay is simple to perform and requires no washing steps.
Components
Component |
Concentration |
100T |
500T |
2500T |
10000T |
Storage Temperature |
Tag1-TSLP protein |
100× |
5μL |
20μL |
100μL |
400μL |
-80℃ |
Tag2- TSLPR protein |
100× |
5μL |
20μL |
100μL |
400μL |
-80℃ |
Anti-Tag1 Eu antibody |
50× |
10μL |
50μL |
250μL |
1000μL |
-80℃ |
Anti-Tag2 Ac antibody |
12.5× |
40μL |
200μL |
1000μL |
4000μL |
-80℃ |
Detection buffer |
10× |
400μL |
2mL |
10mL |
40mL |
-80℃ |
Protocol
1. Reagent Preparation
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1.1 Thaw all reagents at room temperature before use (allow at least 30 min for equilibration). The reaction system for the 384-well shallow plate is 20 μL (reagent volumes are shown in the table below). Calculate the required volume before preparation. The following preparation is for reference only, using 500 reactions as an example.
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Table 1. Reagent Preparation
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Reagent Name |
Preparation |
Volume per Well (μL) |
Detection buffer |
Take 2 mL of 10× Detection buffer, add 18 mL of deionized water, dilute to 1×, and mix well. | - |
Tag1-TSLP protein |
Take 20 μL of Tag1-TSLP protein stock, dilute to 2 mL with 1× Detection buffer, and mix well. | 4 |
Tag2-TSLPR protein |
Take 20 μL of Tag2-TSLPR protein stock, dilute to 2 mL with 1× Detection buffer, and mix well. | 4 |
Mix |
Take 50 μL of Anti-Tag1 Eu antibody stock, add 2.45 mL of 1× Detection buffer, and mix well; Take 200 μL of Anti-Tag2 Ac antibody stock, add 2.3 mL of 1× Detection buffer, and mix well; After dilution, mix Anti-Tag1 Eu antibody and Anti-Tag2 Ac antibody 1:1 and mix well. |
10 |
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1.2 Gradient Dilution of Test Samples
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Using Bosakitug as an example, the dilution buffer is 1× Detection buffer. To reduce matrix interference, it is recommended to use a solution with the same matrix as the test sample. Adjust the dilution based on the actual sample concentration.
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Table 2. Gradient Dilution of Positive Control Bosakitug (adjust as needed)
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|
Final Bosakitug Concentration (nM) |
Preparation Concentration (nM) |
Preparation Method |
① |
1000 |
10000 |
3 μL of 57.5 μM Bosakitug + 14.4 μL of 1× Detection buffer |
② |
333.333 |
3333.333 |
5 μL of ① + 10 μL of 1× Detection buffer |
③ |
111.111 |
1111.111 |
5 μL of ② + 10 μL of 1× Detection buffer |
④ |
37.037 |
370.370 |
5 μL of ③ + 10 μL of 1× Detection buffer |
⑤ |
12.346 |
123.457 |
5 μL of ④ + 10 μL of 1× Detection buffer |
⑥ |
4.115 |
41.152 |
5 μL of ⑤ + 10 μL of 1× Detection buffer |
⑦ |
1.372 |
13.717 |
5 μL of ⑥ + 10 μL of 1× Detection buffer |
⑧ |
0.457 |
4.572 |
5 μL of ⑦ + 10 μL of 1× Detection buffer |
⑨ |
0.152 |
1.524 |
5 μL of ⑧ + 10 μL of 1× Detection buffer |
⑩ |
0.051 |
0.508 |
5 μL of ⑨ + 10 μL of 1× Detection buffer |
⑪ |
0.017 |
0.169 |
5 μL of ⑩ + 10 μL of 1× Detection buffer |
Blank |
0 |
0 |
15 μL of 1× Detection buffer |
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2. Sample Loading and Controls
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2.1 Sample loading order for test wells: 2 μL of test sample, 4 μL of Tag1-TSLP protein working solution, 4 μL of Tag2-TSLPR protein working solution, and 10 μL of mixed Mix, added sequentially to the 384-well shallow plate.
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2.2 Maximum control: 2 μL of 1× Detection buffer diluent, 4 μL of Tag1-TSLP protein working solution, 4 μL of Tag2-TSLPR protein working solution, and 10 μL of mixed Mix, added sequentially to the 384-well shallow plate.
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2.3 Negative control (NC): 10 μL of 1× Detection buffer + 10 μL of Mix.
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After loading all samples, centrifuge, seal with plate film, and incubate at room temperature for 2 hours.
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|
Maximum Control |
Sample |
NC |
Step 1 |
2 μL 1× Detection Buffer (with DMSO) |
2 μL Gradient-diluted test sample |
10 μL of 1× Detection Buffer |
4 μL of Tag1-TSLP protein working solution | |||
Incubate for 10 min | |||
Step 2 |
4 μL of Tag2-TSLPR protein working solution |
||
10 μL of Mix | |||
Step 3 |
Seal with plate film and incubate at room temperature for 2 h |
||
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3.Detection
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Measure using a TR-FRET-compatible microplate reader. Excitation wavelength: 320/340 nm; emission wavelengths: 620 nm and 665 nm.
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[Data Analysis]
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1) Calculate the signal ratio (Ratio): Divide the 665 nm fluorescence signal by the 620 nm signal and multiply by 10,000.
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Ratio = (665/620) × 10,000
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2) Calculate the net signal:
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Net signal = (Std - NC) / NC × 100
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3) Calculate CV (%):
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CV (%) = (Standard Deviation / Mean Ratio) × 100%
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[Example Data]
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The following data is for illustration only and may vary depending on the plate reader used.
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Note: Recommended microplate (384-well, white, shallow-well plate)
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