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【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.
来源
Human储存条件
-80℃
【Composition and Storage Conditions】
Composition
concentration
100T
500T
2500T
10000T
Storage
Tag1-NLRP3 protein
100×
5μL
20μL
100μL
400μL
-80℃
Tag2- NEK7 protein
50×
8μL
40μL
200μL
800μL
-80℃
Anti-Tag1 Eu antibody
100×
5μL
25μL
125μL
500μL
-80℃
Anti-Tag2 Ac antibody
100×
5μL
25μL
125μL
500μL
-80℃
Detection buffer
1×
3mL
15mL
75mL
300mL
-80℃
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
Composition
Configuration
μL/well
Tag1-NLRP3 protein
Take 20 μL of the 100× Tag1-NLRP3 protein, add 1.98 mL of Detection buffer to dilute it to 1× concentration, and mix thoroughly for later use.
4
Tag2- NEK7 protein
Take 40 μL of 50× Tag2- NEK7 protein, add 1.96mL of Detection buffer to dilute it to 1× concentration, and mix thoroughly for later use.
4
Antibody Mix
Take 25 μL of the 100× Anti-Tag1 Eu antibody stock solution, add 2475 μL of Detection buffer to dilute to 2.5 mL, and mix thoroughly; take 25 μL of the 100× Anti-Tag2 Ac antibody stock solution, add 2475 μL of Detection buffer to dilute to 2.5 mL, and mix thoroughly; combine the two solutions in a 1:1 ratio to obtain the Antibody Mix.
10
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
Sample
Blank
Negative control(NC)
2μL
gradient-diluted sample
2μL
Detection buffer
10μL Detection buffer
and 10μL Antibody Mix
4μL Tag1-NLRP3 protein*
Seal the plate wells with a sealing film and incubate at room temperature for 10 minutes.
4μL Tag2-NEK7 protein*
10μL Antibody Mix
Seal the plate wells with a sealing film and incubate at room temperature for 2 hours.
*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).
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