UniOne® TR-FRET Human BAFF/TACI Binding Kit
UniOne® TR-FRET Human BAFF/TACI Binding Kit
Product Details
Product Details
Product Specification
| Host | Human |
| Stability & Storage | -80℃ |
Background
The kit employs homogeneous time-resolved fluorescence (TR-FRET) technology to measure the interaction between Human BAFF and Human TACI. This method enables simple, rapid, and high-throughput screening of inhibitors and antibody blockers.
As shown in the figure, the interaction between BAFF and TACI is detected using an Eu-labeled anti-Tag1 antibody (TR-FRET donor) and an acceptor-labeled Tag2 antibody (TR-FRET acceptor). The binding of BAFF and TACI brings the donor and acceptor antibodies into close proximity, allowing the excitation of the donor antibody to trigger fluorescence resonance energy transfer (FRET) to the acceptor antibody, resulting in a specific 665 nm emission signal. The positive control drug Tabalumab blocks the binding of BAFF and TACI, preventing FRET signal generation. The stronger the inhibitory effect of the screened drug on BAFF and TACI interaction, the lower the signal. The signal intensity is proportional to the degree of BAFF-TACI interaction. No washing steps are required.
Components
组分 |
浓度 |
100T |
500T |
2500T |
10000T |
储存温度 |
Tag1-BAFF protein |
100 × |
5μL |
20μL |
100μL |
400μL |
-80℃ |
Tag2- TACI protein |
100 × |
5μL |
20μL |
100μL |
400μL |
-80℃ |
Tabalumab |
25μM |
5μL |
15μL |
75μL |
300μL |
-80℃ |
Eu-anti-Tag1 |
50 × |
10μL |
50μL |
250μL |
1000μL |
-80℃ |
Ac-anti-Tag2 |
12.5 × |
40μL |
200μL |
1mL |
4mL |
-80℃ |
Detection buffer |
10 × |
400μL |
2mL |
10mL |
40mL |
-80℃ |
注意：所有首次解冻后立即分装，并按建议储存温度保存，避免稀释后储存及反复冻融。
Protocol
1. Reagent Preparation
1.1 Thaw all reagents at room temperature before use (allow at least 30 minutes for equilibration). The reaction system for the 384-well plate is 20μL (reagent volumes per reaction are shown in the table below). Calculate the required volume before preparation and prepare accordingly. The following preparation is for reference only, using 500 reactions as an example.
Table 1. Reagent Preparation
Reagent Name |
Preparation |
Volume per Well (μL) |
Detection buffer |
Take 2 mL of 10× Detection buffer and add 18 mL of deionized water to dilute to 1×. Mix well and set aside. |
- |
Tag1-BAFF protein |
Take 20 μL of 100× Tag1-BAFF protein stock solution and add to 1.98 mL of 1× Detection buffer to dilute to 1×. Mix well and set aside. |
4μL |
Tag2-TACI protein |
Take 20 μL of 100× Tag2-TACI protein stock solution and add to 1.98 mL of 1× Detection buffer to dilute to 1×. Mix well and set aside. |
4μL |
|
Detection Reagent Mix
|
Take 50 μL of 50× Eu-anti-Tag1 stock solution and add to 2.45 mL of 1× Detection buffer to dilute to 2.5 mL. Mix well. Take 200 μL of 12.5× Ac-anti-Tag2 stock solution and add to 2.30 mL of 1× Detection buffer to dilute to 2.5 mL. Mix well. Mix the two solutions at a 1:1 ratio to prepare the Detection Mix. |
10μL |
1.2 Gradient Dilution of Test Samples
Taking Tabalumab as an example, the dilution buffer is 1× Detection buffer. To minimize matrix interference effects, it is recommended to use a solution with the same matrix as the test sample for dilution. Adjustments should be made based on the actual concentration of the sample.
Table 2. Gradient Dilution of Positive Control Tabalumab (Adjust as Needed)
|
Tabalumab Preparation Concentration (nM) |
Tabalumab Final Concentration (nM) |
Preparation Method |
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① |
2500 |
250 |
3μL 25μM stock solution + 27μL 1× Detection buffer |
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2. Sample Loading and Controls 2.1 Sample Well: Add 2μL of test sample (gradient dilution), 4μL Tag1-BAFF protein working solution, 4μL Tag2-TACI protein working solution, and 10μL detection reagent Mix sequentially into a 384-well shallow plate. 2.2 Maximum Signal Control: Add 2μL Detection buffer, 4μL Tag1-BAFF protein working solution, 4μL Tag2-TACI protein working solution, and 10μL detection reagent Mix sequentially into a 384-well shallow plate. 2.3 Negative Control: Add 10μL Detection buffer and 10μL detection reagent Mix sequentially into a 384-well shallow plate. After loading all samples, centrifuge, seal with plate film, and incubate at room temperature for 2 hours.
3、Detection Perform detection on a TR-FRET-compatible microplate reader. The excitation wavelength is 320/340 nm, and the emission wavelengths are 620 nm and 665 nm. 【Result Calculation】 1) Calculate the signal value (Ratio): Divide the 665 nm fluorescence signal by the 620 nm fluorescence signal and multiply by 10,000. Ratio = (665/620) × 10,000 2) Calculate the Net signal based on the signal value: Net signal = (Std - NC)/NC × 100 3) Calculate CV (%): CV (%) = Standard Deviation/Mean Ratio × 100%
【Data Example】 The following data is for illustrative purposes only and cannot replace the data obtained in experiments. Results may vary depending on the plate reader.
Note: Recommended microplate (384-well plate, white, shallow well) | |||||||||||||||||||||
