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UniOne® TR-FRET Human TNF-α TNFR1 Binding Kit

UniOne® TR-FRET Human TNF-α TNFR1 Binding Kit

Catalog Number: UA086007 Brand: UA BIOSCIENCE
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Regular price $2,180 USD
Regular price Sale price $2,180 USD
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Product Details

Product Specification


Host Human
Stability & Storage

-80℃

Background

The kit utilizes homogeneous time-resolved fluorescence technology (TR-FRET) to measure the interaction between TNF-α and TNFR1. This method enables simple and rapid high-throughput screening of inhibitors and antibody blockers.

As shown in the figure, the interaction between TNF-α and TNFR1 is detected using a Eu-labeled anti-Tag1 antibody (TR-FRET donor) and an Ac-labeled anti-Tag2 antibody (TR-FRET acceptor). The binding of TNF-α to TNFR1 brings the donor and acceptor antibodies into close proximity, allowing fluorescence resonance energy transfer (FRET) from the donor to the acceptor, resulting in a specific emission signal at 665 nm. The positive control drug Adalimumab blocks the binding of TNF-α to TNFR1, preventing FRET signal generation. The stronger the inhibitory effect of the screened drug on the TNF-α/TNFR1 interaction, the lower the signal. This specific signal is proportional to the extent of TNF-α/TNFR1 interaction. The homogeneous assay is simple to perform and requires no washing steps.

Components

Component

Concentration

100T

500T

2500T

10000T

Storage Temperature

Tag1-TNF-α protein

100 ×

5μL

20μL

100μL

400μL

-80℃

Tag2- TNFR1 protein

100 ×

5μL

20μL

100μL

400μL

-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℃


Note: Aliquot immediately after initial thawing and store at recommended temperatures. Avoid storage after dilution and repeated freeze-thaw cycles.

Protocol

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1. Reagent Preparation

1.1 Thaw all reagents at room temperature before use (equilibrate for at least 30 min). The reaction system in 384-well plates is 20μL (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.

Table 1. Reagent Preparation

Reagent Name

Preparation

Volume per Well (μL)

Detection buffer

Add 2mL of 10× Detection buffer to 18 mL deionized water, dilute to 1×, and mix well.

-

Tag1-TNF-α protein

Add 20μL of 100× Tag1-TNFα protein stock to 1.98 mL 1× Detection buffer, dilute to 1×, and mix well.

4

Tag2-TNFR1 protein

Add 20μL of 100× Tag2-TNFR1 protein stock to 1.98 mL 1× Detection buffer, dilute to 1×, and mix well.

4

Detection Mix

Add 50μL of 50× Eu-anti-Tag1 stock to 2.45 mL 1× Detection buffer, dilute to 2.5 mL, and mix well;

Add 200μL of 12.5× Ac-anti-Tag2 stock to 2.30 mL 1× Detection buffer, dilute to 2.5 mL, and mix well;

Mix the two solutions at 1:1 ratio to prepare the Detection Mix.

10


1.2 Sample serial dilution

Take Adalimumab as an example. The diluent is 1× Detection buffer. To minimize matrix effects, it is recommended to use a solution with the same matrix as the sample. Adjust the dilution according to the actual sample concentration.

Table 2. Adalimumab serial dilution (adjust as needed)

Adalimumab Final Conc. (nM)

Adalimumab Prep. Conc. (nM)

Preparation Method

1000.00

10000.00

1μL 375μM stock + 36.5μL 1× Detection buffer

400.000

4000.000

10μL +15μL 1× Detection buffer

160.000

1600.000

10μL +15μL 1× Detection buffer

64.000

640.000

10μL +15μL 1× Detection buffer

25.600

256.000

10μL +15μL 1× Detection buffer

10.240

102.400

10μL +15μL 1× Detection buffer

4.096

40.960

10μL +15μL 1× Detection buffer

1.638

16.384

10μL +15μL 1× Detection buffer

0.655

6.554

10μL +15μL 1× Detection buffer

0.262

2.621

10μL +15μL 1× Detection buffer

0.105

1.049

10μL +15μL 1× Detection buffer

Blank

0

0

15μL 1× Detection buffer


2. Sample Loading and Controls

2.1 Sample wells: Add 2μL of sample (serial dilution), 4μL Tag1-TNF-α protein working solution, 4μL Tag2-TNFR1 protein working solution, and 10μL Detection Mix sequentially to 384-well plates.

2.2 Max signal control: Add 2μL Detection buffer, 4μL Tag1-TNF-α protein working solution, 4μL Tag2-TNFR1 protein working solution, and 10μL Detection Mix sequentially to 384-well plates.

2.3 Negative control (NC): Add 10μL Detection buffer and 10μL Detection Mix sequentially to 384-well plates.

After loading all samples, centrifuge, seal with plate seal, and incubate at room temperature for 2 hours.

 

Sample

Max Signal Control

Negative Control (NC)

Step 1

2μL sample

2μL 1×Detection buffer

10μL 1×Detection buffer

4μL Tag1-TNF-α protein working solution

Incubate at RT for 10 min

Step 2

4μL Tag2-TNFR1 protein working solution

10μL Detection Mix

Seal plate and incubate at RT for 2h


3. Detection

Read plates using a TR-FRET compatible microplate reader. Excitation wavelength: 320/340 nm; emission wavelengths: 620 nm and 665 nm.

[Data Analysis]

1) Calculate signal ratio: Divide 665 nm fluorescence signal by 620 nm fluorescence signal and multiply by 10000.

Ratio = (665/620) ×10000

2) Calculate Net signal based on ratio:

Net signal = (Std-NC)/NC×100

3) Calculate CV (%):

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

[Example Data]

The following data is for reference only and may vary depending on the plate reader used.

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

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