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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4.8 ★★★★★
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Product Reviews
R
Verified Purchase
Ruth Ann Burt
Lowell, US
★★★★★ 5
Great book
Format: Kindle
I absolutely feel in love with all 4 characters!!! The bedroom scenes were 🌋🌡🔥🔥🔥. I couldn't put this book down!!! I'm hooked for the whole series Book 2 here I come!!!!! Its a fun easy book and story to read!!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on October 4, 2024
D
Verified Purchase
Danyelle
Belleville, US
★★★★★ 4
Fun with a late blooming omega
Format: Kindle
I like this book. The story is fun, cute, and sexy. There's just a little drama, some excellent, steamy scenes, and a fairly good relationship building storyline. I especially like how all the main characters are a bit older than the usual 20 somethings I tend to see in this kind of book. Having said that, I wish there were more descriptions of the places, as well as the food in the fancy restaurant. I enjoyed the cocktails at the club, so I missed that kind of detail when Gray took Madison on a dinner date. I also wish there had been more interaction between Lucas and Madison, and Lucas and Rian. It felt a bit lopsided, with a focus on Rian, Madison, and Gray. I wish it had been proofread - there are a lot of typos, but nothing too distracting.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on September 12, 2022
J
Verified Purchase
Jennifer G
Alexandria, US
★★★★★ 3
Madison Deserved Better
Format: Kindle
Madison was a beta...except she wasn't any longer. She was a late presenting Omega. And she was struggling. She was tall and thin, not tiny and curvy. She was opinionated. She was everything an Omega was not. After suffering through her first heat, her friends took her to Ardor, a club where Omegas came to safely find Alphas. She's not expecting much but then she connects with a sexy beta. And when she meets his Alphas, they set her body on fire. Maybe, she's found her no-strings-attached heat pack. Maybe, she's found something more. I could not connect with the characters in this book, so their story never resonated with me. And there was no love story; there was sex. Grey made it clear from the beginning that he had a true love and it was his beta boy, Rian. He went so far as to reassure Rian “Say the word, I’ll never touch her again. Lucas can put the babies in her. I only need you, beta boy”. So, Madison was there for babies, no emotions needed. Nice. No, thank you. I want the Omega to be the center of their world, not an incubator. Lucas and Rian weren't any better. After her heat, they let her leave. Not one of them made her feel valued. No one gave her a reason to stay or even offered a cuddle. And the sex didn't even come across as mind-blowing. Madison deserved better.
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Reviewed in the United States on March 11, 2025
O
Verified Purchase
Oregon BookWorm
Chelsea, US
★★★★★ 5
No breakup, very sweet, instalove
Format: Kindle
Omegaverse and doesn't disappoint! Sweet guys, newly Omega FMC. The boyfriends are boyfriends. What's not to love? No angst, no breakup.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on February 23, 2025
F
Verified Purchase
ForTheLOVEofBooks
Carnegie, US
★★★★★ 4
Pretty Darn Good
Format: Kindle
So I’ve been on a omega kick and this definitely hit the spot. Madison was frustrating at times with how she acted towards Lucas, Gray, and Rian. It was like she said towards the end, she didn’t believe she deserved nice things. It would have been nice to hear from her best friends again. They kind of were there in the beginning and the gone except for mention of text messages received from them. I feel like her friends would have been great help in encouraging Madison to go with the pack and never give Brent another chance because he was toxic. I loved Rian. His personality was awesome. His humor. His ability to make Madison comfortable whenever she was feeling overwhelmed. And the fact he fell for her and she fell for him first. They are cute together. I do feel like Lucas was the odd man out though. Like Lucas didn’t develop as much of a relationship with Madison. I would have really liked to see more development in the relationship between them. It was also the same with him and Rian. There is really no relationship displayed. Most of the relationship being displayed is between Rian and Gray. Nevertheless, I loved reading about the dynamic that came to fruition during the entirety of this story. Madison finally got her happiness. And Brent finally got punched in the face. Everyone got exactly what they deserve.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on September 6, 2022

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