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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 DDB1/CRBN complex and ITK. This method provides a high-throughput approach to detect the small molecules capable of mediating the interaction between the DDB1/CRBN complex and ITK simply and rapidly.
As shown in the figure below, the interaction between DDB1/CRBN and ITK 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 BSJ-05-037 mediates the interaction between DDB1/CRBN and ITK, the donor antibody 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 BSJ-05-037, DDB1/CRBN and ITK. 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-CRBN protein
100×
5μL
20μL
100μL
400μL
-80℃
Tag2-ITK protein
78×
6μL
26μL
130μL
520μL
-80℃
BSJ-05-037
500μM
5μL
10μL
50μL
200μL
-80℃
Anti-Tag1 Eu antibody
50×
10μL
50μL
250μL
1000μL
-80℃
Anti-Tag2 Ac antibody
12.5×
40μL
200μL
1000μL
4mL
-80℃
Detection buffer
10×
400μL
2mL
10mL
40mL
-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
配置
μL/well
Detection buffer
Add 2 mL of 10× Detection buffer to 18 mL of deionized water, dilute to 1× Detection , and mix thoroughly for later use.
-
Tag1-CRBN protein
Take 20 μL of the 100× Tag1-CRBN protein stock solution, add 1.98 mL of 1× Detection buffer to dilute it to 1× concentration, and mix thoroughly for later use.
4
Tag2-ITK protein
Take 26 μL of 78× Tag2-ITK protein, add it to 2.002 mL of 1× Detection buffer to dilute to 1× concentration, and mix thoroughly for later use.
4
Antibody Mix
Take 50 μL of the 50× Eu-anti-Tag1 stock solution, add 2450 μL of 1× Detection buffer to dilute to 2.5 mL, and mix thoroughly; take 200 μL of the 12.5× Ac-anti-Tag2 stock solution, add 2300 μL of 1× 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
BSJ-05-037 as an example, the dilution solution is 1× 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. BSJ-05-037 gradient dilution（Adjust according to the actual situation）
BSJ-05-037 final concentration（nM）
BSJ-05-037 preparation concentration（nM）
Dilution
①
1000
10000
2μL 500 μM BSJ-05-037 +98μL 1× Detection buffer
②
333.33
3333.33
30μL ① +60μL 1× Detection buffer
③
111.11
1111.11
30μL ② +60μL 1× Detection buffer
④
37.04
370.37
30μL ③ +60μL 1× Detection buffer
⑤
12.35
123.46
30μL ④ +60μL 1× Detection buffer
⑥
4.12
41.15
30μL ⑤ +60μL 1× Detection buffer
⑦
1.37
13.72
30μL ⑥ +60μL 1× Detection buffer
⑧
0.46
4.57
30μL ⑦+60μL 1× Detection buffer
⑨
0.15
1.52
30μL ⑧+60μL 1× Detection buffer
⑩
0.05
0.51
30μL ⑨+60μL 1× Detection buffer
Blank
0
0
60μL 1× Detection buffer
2、Sample and control
2.1 Sample：Add 4 μL of Tag1-CRBN protein working solution, 4 μL of Tag2-ITK 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 BSJ-05-037: 4 μL of Tag1-CRBN protein working solution, 4 μL of Tag2- ITK protein working solution, 2 μL of gradient-diluted BSJ-05-037, and 10 μL of the mixed Antibody Mix sequentially into the 384-well plate.
2.3 Blank：4 μL of Tag1-CRBN protein working solution, 4 μL of Tag2-ITK 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 for 2 hours.
Table 3. Sample and control spotting process
DEG-35
Sample
Blank
Negative control(NC)
2μL
gradient-diluted BSJ-05-037
2μL
gradient-diluted sample
2μL
1× Detection buffer
10μL 1× Detection buffer
and 10μL Antibody Mix
4μL Tag1-CRBN protein
4μL Tag2-ITK protein
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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