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UniOne^® TR-FRET Human BRD4/AR RIPTAC Binding Kit

Datasheet COA

价格￥4,380.00 （供应商现货： 3-5个工作日）

货号 UA086148

规格

100T500T2500T10000T

数量

产品介绍

产品介绍评论(0)

背景介绍

【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 the BRD4 (BD1/BD2) and the LBD of human androgen receptor (AR LBD). This method provides a high-throughput approach to detect the small molecules capable of mediating the interaction between BRD4 and AR LBD simply and rapidly .
As shown in the figure below, the interaction between BRD4 and AR LBD was detected by an Eu-labeled anti-Tag1 antibody (TR-FRET donor) and an Ac-labeled anti-Tag2 antibody (TR-FRET receptor). Since the molecular adhesive HLD0915 mediates the interaction between BRD4 and AR LBD, 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. This specific signal is proportional to the degree of interaction between molecular adhesive, BRD4 and AR LBD. This homogeneous assay is simple to perform and requires no washing steps.

产品规格

来源

Human
储存条件

-80℃

产品组分

【Composition and Storage Conditions】

Composition	concentration	100T	500T	2500T	10000T	Storage
Tag1-AR LBD	50×	8μL	40μL	200μL	800μL	-80℃
Tag2-BRD4 protein	100×	5μL	20μL	100μL	400μL	-80℃
HLD0915	1 mM	5μL	10μL	50μL	200μL	-80℃
Anti-Tag1 Eu antibody	100×	5μL	25μL	125μL	500μL	-80℃
Anti-Tag2 Ac antibody	100×	5μL	25μL	125μL	500μL	-80℃
Detection buffer	1×	4mL	20mL	100mL	400mL	-80℃

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

操作步骤

【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-AR LBD	Take 40 μL of the 50× Tag1-AR LBD stock solution, add 1.96 mL of Detection buffer to dilute it to 1× concentration, and mix thoroughly for later use.	4
Tag2-BRD4 protein	Take 20 μL of 100× Tag2-BRD4 protein protein, add it to 1.98 mL of Detection buffer to dilute 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

HLD0915 as an example, the dilution solution is Detection buffer. To minimize interference from matrix effects, it is recommended to use a solution with the same matrix composition as the sample being tested; additionally, the sample concentration should be adjusted according to its actual situation.

Table 2. HLD0915 gradient dilution（Adjust according to the actual situation）

	HLD0915 final concentration（nM）	HLD0915 preparation concentration（nM）	Dilution
①	8000.00	80000.00	2.5μL 1 mM HLD0915 +28.75μL Detection buffer
②	2666.67	26666.67	10μL ① +20μL Detection buffer
③	888.89	8888.89	10μL ② +20μL Detection buffer
④	296.30	2962.96	10μL ③ +20μL Detection buffer
⑤	98.77	987.65	10μL ④ +20μL Detection buffer
⑥	32.92	329.22	10μL ⑤ +20μL Detection buffer
⑦	10.97	109.74	10μL ⑥ +20μL Detection buffer
⑧	3.66	36.58	10μL ⑦+20μL Detection buffer
⑨	1.22	12.19	10μL ⑧+20μL Detection buffer
⑩	0.41	4.06	10μL ⑨+20μL Detection buffer
Blank	0	0	20μL Detection buffer

2、Sample and control

2.1 Sample：Add 4 μL of Tag1-AR LBD working solution, 4 μL of Tag2-BRD4 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 HLD0915: 4 μL of Tag1-AR LBD working solution, 4 μL of Tag2-BRD4 protein working solution, 2 μL of gradient-diluted HLD0915, and 10 μL of the mixed Antibody Mix sequentially into the 384-well plate.

2.3 Blank：4 μL of Tag1-AR LBD working solution, 4 μL of Tag2-BRD4 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 more than 6hours.

Table 3. Sample and control spotting process

HLD0915	Sample	Blank	Negative control(NC)
2μL gradient-diluted HLD0915	2μL gradient-diluted sample	2μL Detection buffer	10μL Detection buffer and 10μL Antibody Mix
4μL Tag1-AR LBD
4μL Tag2-BRD4 protein
10μL Antibody Mix
Seal the plate wells with a sealing film and incubate at room temperature more than 6 hours.

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).

UniOne® TR-FRET Human BRD4/AR RIPTAC Binding Kit

来源

储存条件

UniOne^® TR-FRET Human BRD4/AR RIPTAC Binding Kit