SKU: 53650331176

UniOne® TR-FRET Human VCB(VHL Complex)Binding Kit

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Description

UniOne® TR-FRET Human VCB(VHL Complex)Binding KitProduct Specification Host Human Stability & Storage 80 Background The reagent kit employs homogeneous time resolved fluorescence technology (TR FRET) for measuring the interaction between Human VCB (VHL Elongin C Elongin B Complex) and Human HIF 1. The Tag2 Tracer is a fluorescently labeled HIF 1 peptide fragment. This method enables simple and rapid high throughput screening of inhibitors and antibody blockers. As shown in the figure, the

Product Specification


Host Human
Stability & Storage

-80℃

Background

The reagent kit employs homogeneous time-resolved fluorescence technology (TR-FRET) for measuring the interaction between Human VCB (VHL/Elongin-C/Elongin-B Complex) and Human HIF-1α. The Tag2-Tracer is a fluorescently labeled HIF-1α peptide fragment. This method enables simple and rapid high-throughput screening of inhibitors and antibody blockers.

As shown in the figure, the interaction between VCB and Tracer is detected using a Eu-labeled anti-Tag1 antibody (TR-FRET donor) and an Acceptor-labeled SA (TR-FRET acceptor). Since the binding of VCB and Tracer brings the donor antibody and acceptor antibody into proximity, excitation of the donor antibody triggers fluorescence resonance energy transfer (FRET) to the acceptor antibody, resulting in a specific emission signal at 665 nm. VH032 can block the binding of VCB and Tracer, preventing the generation of FRET signals. The stronger the blocking effect of the screened drug on the VCB-Tracer interaction, the lower the signal. The signal is proportional to the degree of interaction between VCB and Tracer. No washing steps are required.

Components

组分

浓度

100T

500T

2500T

10000T

储存温度

Tag1-VCB protein

100 ×

5μL

20μL

100μL

400μL

-80℃

Tag2-Tracer

100 ×

5μL

20μL

100μL

400μL

-80℃

Eu-anti-Tag1

50 ×

10μL

50μL

250μL

1000μL

-80℃

SA-Ac

100 ×

5μL

25μL

125μL

500μL

-80℃

Detection buffer

1 ×

3mL

14mL

70mL

240mL

-80℃


Protocol

1. Reagent Preparation

1.1 Before use, thaw all reagents at room temperature (room temperature equilibration for at least 30 min). The 384-well shallow well plate reaction system volume is 20 μL (reagent volumes for the reaction system are shown in the table); calculate the required volume before preparation and prepare as needed; the following preparation is for reference only, taking 500 times as an example.

Table 1. Reagent Preparation

Reagent Name

Preparation

Volume per Detection Well μL

Tag1-VCB protein

Take 20 μL 100 × Tag1-VCB protein stock solution, add to 1.98 mL 1×Detection buffer to dilute to 1×, mix and set aside.

4μL

Tag2-Tracer

Take 20 μL 100 × Tag2-Tracer stock solution, add to 1.98 mL 1×Detection buffer to dilute to 1×, mix and set aside.

4μL

Detection Reagent Mix

 

Take 50 μL 50× Eu-anti-Tag1 stock solution, add 2.45 mL 1×Detection buffer to dilute to 2.5 mL, mix;

Take 25 μL 100× SA-Ac stock solution, add 1×Detection buffer 2.475 mL to dilute to 2.5 mL, mix;

Mix both 1:1, this is Detection Mix.

10μL


 

1.2 Gradient Dilution of Test Samples

Taking VH032 as an example, the diluent is 1× Detection buffer. To reduce the interference of matrix effects, it is recommended to use a solution with the same matrix as the test sample for dilution; and adjust the test samples according to actual concentration.

Table 2. Positive Drug VH032 Gradient Dilution (Adjust According to Actual Situation)

 

VH032 Prepared Concentration (nM)

VH032 Final Concentration (nM)

Preparation Method

30,000.0

300,000.0

2μL 3mM Stock Solution + 18μL 1× Detection buffer

10000.0

100000.0

10μL ① +20μL 1× Detection buffer

3333.3

33333.3

10μL ② +20μL 1× Detection buffer

1111.1

11111.1

10μL ③ +20μL 1× Detection buffer

370.4

3703.7

10μL ④ +20μL 1× Detection buffer

123.5

1234.6

10μL ⑤ +20μL 1× Detection buffer

41.2

411.5

10μL ⑥ +20μL 1× Detection buffer

13.7

137.2

10μL ⑦ +20μL 1× Detection buffer

4.6

45.7

10μL ⑧ +20μL 1× Detection buffer

Blank

0.0

0

20μL 1× Detection buffer


 

 

2. Sample Addition and Controls

2.1 Sample Well (Sample): Add 2 μL test sample (gradient dilution), 4 μL Tag1-VCB protein working solution, 4 μL Tag2-Tracer working solution, 10 μL Detection Reagent Mix sequentially into the 384-well shallow well plate.

2.2 Maximum Signal Control: Add 2 μL Detection buffer, 4 μL Tag1-VCB protein working solution, 4 μL Tag2-Tracer working solution, 10 μL Detection Reagent Mix sequentially into the 384-well shallow well plate.

2.3 Negative Control (Negative control): Add 10 μL Detection buffer, 10 μL Detection Reagent Mix sequentially into the 384-well shallow well plate.

After adding all samples, centrifuge, seal the plate film, and incubate at room temperature for 2 hours.

 

Sample (Sample)

Maximum Signal Control

Negative Control (NC)

Step 1

2μL Test Sample

2μL 1×Detection buffer

10μL 1×Detection buffer

4μL Tag1-VCB protein Working Solution

Incubate at Room Temperature 10 min

Step 2

4μL Tag2-Tracer Working Solution

10μL Detection Reagent Mix

Seal plate with film, incubate at room temperature for 2h


 

 

3.Detection

Detect on a microplate reader compatible with TR-FRET. Excitation light is 320/340 nm, detect emission wavelengths of 620 nm and 665 nm.

[Result Calculation]

1) Calculate Signal Value (Ratio): Fluorescence signal at 665 nm divided by fluorescence signal at 620 nm, then multiplied by 10000.

Ratio = (665/620) ×10000

2) Calculate Net signal based on signal value:

Net signal = (Std-NC)/NC×100

3) Calculate CV (%):

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

 

[Data Example]

The data below cannot replace data obtained from experiments, it is for demonstration purposes only; results may vary depending on the plate reader instrument.

 

Note: Recommended Microplate (384-well plate, White, Shallow Well)

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SKU: 53650331176

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