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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 the BRD4 (BD2) and the H4 peptide. This method provides a high-throughput approach to detect the small molecules capable of mediating the interaction between BRD4 (BD2) and H4 peptide simply and rapidly.
As shown in the figure below, the interaction between BRD4 (BD2) and H4 peptide was detected by an Eu-labeled anti-Tag1 antibody (TR-FRET donor) and an Ac-labeled anti-Tag2 antibody (TR-FRET receptor). 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. Since the molecular (+) JQ-1 can block the interaction between BRD4 (BD2) and H4 peptide, the FRET signal is reduced. This specific signal is proportional to the degree of interaction between BRD4 (BD2) and H4 peptide. This homogeneous assay is simple to perform and requires no washing steps.
来源
Human储存条件
-80℃
【Composition and Storage Conditions】
Composition
concentration
100T
500T
2500T
10000T
Storage
Tag1-BRD4 (BD2)
100×
5μL
20μL
100μL
400μL
-80℃
Tag2-H4 peptide
100×
5μL
20μL
100μL
400μL
-80℃
Anti-Tag1 Eu antibody
100×
5μL
25μL
125μL
500μL
-80℃
Anti-Tag2 Ac antibody
12.5×
40μL
200μL
1mL
4mL
-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.
【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-BRD4 (BD2)
Take 20 μL of the 100× Tag1-BRD4 (BD2) stock solution, add 1.98 mL of Detection buffer to dilute it to 1× concentration, and mix thoroughly for later use.
4
Tag2-H4 peptide
Take 20 μL of 100× Tag2-H4 peptide, 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 200 μL of the 12.5× Anti-Tag2 Ac antibody stock solution, add 2300 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
(+) JQ-1 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. (+) JQ-1 gradient dilution（Adjust according to the actual situation）
(+) JQ-1 final concentration（nM）
(+) JQ-1 preparation concentration（nM）
Dilution
①
300
3000
0.5μL 500 μM (+) JQ1 + 82.85μL Detection buffer
②
100.00
1000.00
10μL ① +20μL Detection buffer
③
33.33
333.33
10μL ② +20μL Detection buffer
④
11.11
111.11
10μL ③ +20μL Detection buffer
⑤
3.70
37.04
10μL ④ +20μL Detection buffer
⑥
1.23
12.35
10μL ⑤ +20μL Detection buffer
⑦
0.41
4.12
10μL ⑥ +20μL Detection buffer
⑧
0.14
1.37
10μL ⑦+20μL Detection buffer
⑨
0.05
0.46
10μL ⑧+20μL Detection buffer
⑩
0.02
0.15
10μL ⑨+20μL Detection buffer
Blank
0
0
20μL Detection buffer
2、Sample and control
2.1 Sample：Add 4 μL of Tag1-BRD4 (BD2) working solution, 4 μL of Tag2-H4 peptide 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 (+) JQ-1 : 4 μL of Tag1-BRD4 (BD2) working solution, 4 μL of Tag2-H4 peptide working solution, 2 μL of gradient-diluted (+) JQ-1 , and 10 μL of the mixed Antibody Mix sequentially into the 384-well plate.
2.3 Blank：4 μL of Tag1-BRD4 (BD2) working solution, 4 μL of Tag2-H4 peptide 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
(+)JQ-1
Sample
Blank
Negative control(NC)
2μL
gradient-diluted (+) JQ-1
2μL
gradient-diluted sample
2μL
Detection buffer
10μL Detection buffer
and 10μL Antibody Mix
4μL Tag1-BRD4 (BD2)
Incubate at room temperature for 10min
4μL Tag2-H4 peptide
10μL Antibody Mix
Seal the plate wells with a sealing film and incubate at room temperature for 2 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).
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