UniOne® TR-FRET Human NEK7/NLRP3 Binding Kit

【ASSAY PRINCIPLE】

The kit is based on homogeneous time-resolved fluorescence technology (TR-FRET) technology，which is used to assess the molecular-mediated interaction between human NEK7 and human NLRP3. This method provides a high-throughput approach to detect the small molecules capable of mediating the interaction between NEK7 and NLRP3 simply and rapidly.

As shown in the figure below, the interaction between NEK7 and NLRP3 was detected by an Eu-labeled anti-Tag1 antibody (TR-FRET donor) and an Ac-labeled anti-Tag2 antibody (TR-FRET receptor). Since NEK7 binds to NLRP3, the donor is in close proximity to the receptor antibody. Excitation of the donor antibody triggers fluorescence resonance energy transfer (FRET) to the receptor antibody, resulting in specific emission of a signal at a wavelength of 665 nm from the receptor antibody. The screened compounds block the binding of NEK7 to NLRP3, preventing FRET signal generation. The stronger the compound's ability to inhibit the interaction, the lower the signal intensity. The signal intensity is directly proportional to the extent of NEK7-NLRP3 interaction. No washing step is required.

【Composition and Storage Conditions】

Note: Aliquot into appropriate volumes, store at the recommended temperature and avoid repeated freeze-thaw cycle.

【Process and Operations】

1、Reagent Preparation

1.1 Compositions equilibration

Before use, dissolve all reagents at room temperature (allow at least 30 minutes for temperature equilibration). For the 384-well plate assay, use 20 μL of reaction mixture (the reagent quantities are listed in the table below). Calculate the required volume prior to preparation accordingly. The following preparation steps are provided for reference only, using a 500-well plate as an example.

Table 1. Reagent Preparation

1.2 Gradient dilution of the sample

Prepare test samples using 1× Detection Buffer. If the sample stock solution is prepared in DMSO, it is recommended to keep the DMSO concentration consistent across all reaction systems. Since the Detection Buffer does not contain DMSO, add DMSO to the 1× Detection Buffer according to the sample dilution ratio, with a maximum final concentration of 2%.

2、Sample and control

2.1 Sample：Add 4 μL of Tag1-NLRP3 protein working solution, 4 μL of Tag2-NEK7 protein working solution, 2 μL of the gradient-diluted sample , and 10 μL of the mixed Antibody Mix sequentially into the 384-well plate.

2.2 Blank：4 μL of Tag1-NLRP3 protein working solution, 4 μL of Tag2-NEK7 protein working solution, 2 μL of Detection buffer, and 10 μL of the mixed Antibody Mix sequentially into the 384-well plate.

2.3 Negative control：10μL Detection buffer, and 10 μL of the mixed Antibody Mix sequentially into the 384-well plate.

After adding all samples, centrifuge, seal with a cover membrane, and incubate at room temperature for 2 hours.

Table 3. Sample and control spotting process

*Adjust the compound addition sequence according to the inhibition mechanism of test samples.

3、Read Data

The detection was performed on a TR-FRET microplate reader. The excitation wavelength was 320/340 nm, and the emission wavelengths at 620 nm and 665 nm were detected.

【DATA REDUCTION 】

1) Ratio：

Ratio = (665/620) ×10000

2) Net signal：

Net signal = (Std-NC)/NC×100

Std：the ratio of sample and control

NC: the ratio of negative control

3) CV（%）：

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

【Data Example】

The following data cannot replace experimental results obtained in experiments; they are provided solely for illustrative purposes, and outcomes may vary depending on the plate reader used. 

Note: Recommended microplate (384-well plate, white, shallow wells).