UniOne® TR-FRET Human TNF-α/TNFR2 Binding Kit

This kit employsHomogeneous Time-Resolved Fluorescence (TR-FRET) technology to measure the interactionbetween TNF-α and TNFR2. The method enables simple and rapid high-throughputscreening of inhibitors and antibody blockers.

As shown in thefigure, the TNF-α/TNFR2 interaction is detected using an Eu-labeled anti-Tag1antibody (TR-FRET donor) and an Acceptor-labeled TNF-α protein (TR-FRETacceptor). Binding of TNF-α to TNFR2 brings the donor antibody into proximitywith the acceptor protein. Excitation of the donor antibody triggersFluorescence Resonance Energy Transfer (FRET) to the acceptor, resulting inspecific emission at 665 nm. This specific signal is proportional to the levelof TNF-α/TNFR2 interaction. This homogeneous assay is simple to perform andrequires no wash steps.

* Avoid repeated freeze-thaw cycles. Aliquot after first thaw and store according to conditions indicated. Shelf life is one year.

1Reagent Preparation

Thaw all reagents to room temperature before use (equilibrate at room temperature for at least30min).384-well low-volume plate reaction volume is20μL (reagent volumes for the reaction system are shown in the table). Calculate the required volume for the experiment before preparation and prepare as needed; the following preparation is for reference only, using500reactions as an example.

1.1 Preparation of Buffer (1×)

2mL Detection buffer (10×)add18mLsterile water, mix well and set aside;

Table2. Reagent Preparation and Volumes

1.2 Serial Dilution of Test Samples

Prepare test samples using1×Detection buffer. If the sample stock is inDMSO, it is recommended to maintain a consistentDMSO concentration in the system,(the detection buffer does not containDMSO; please addDMSOto the1×Detection bufferaccording to the dilution of the standard or test sample), with a maximum concentration not exceeding2%.

2 Sample Addition and Controls

2.1 Addition order for experimental wells:2μL test sample,8μL Tag1- TNFR2 proteinworking solution,5μL TNFΑ-01A protein protein,5μL Anti-Tag1 Eu dye antibody, sequentially added into the384-well low-volume plate.

2.2 Minimum control well:10μL 1× Detection buffer (containingDMSO),5μL TNFΑ-01A protein,5μL Anti-Tag1 Eu dye antibody.

2.3 Maximum control well:2μL 1× Detection buffer (containingDMSO),8μL Tag1- TNFR2 protein,5μL TNFΑ-01A protein,5μL Anti-Tag1 Eu dye antibody.

2.4 Eu only:15μL 1× buffer plus5μLdilutedAnti-Tag1 Eu dye antibody.

Table4. Sample Addition and Controls

*For negative controls and quality control, it is recommended to usea solution with the same matrix as the test sample to replace1×Detection buffer.

*Ensure consistentDMSOor other matrix concentrations across all detection wells.

3.Detection

Detect using a microplate reader compatible withTR-FRET (excitation wavelength at320nm, emission wavelengths at620nm and665nm).

[Result Calculation]

1. Calculate signal value:665nmfluorescence signal divided by620nmfluorescence signal, then multiplied by10000, i.e.,

Signal Value= (665/620)*10000

2. Calculate the δ value based on the signal value:

δ Value= (Std-NC)/NC*100

3.CalculateCV (%)

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

[Data Example]

The following data cannot replace data obtained from actual experiments and is provided for illustration only. Results may vary depending on theTR-FRETcompatible instrument.