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UniOne^® TR-FRET Human KRAS[G12C]/CypA Ternary Binding Kit

Datasheet COA

价格￥13,080.00 （供应商现货： 3-5个工作日）

货号 UA086152

规格

500T100T2500T10000T

数量

产品介绍

产品介绍评论(0)

背景介绍

The kit is based on homogeneous time-resolved fluorescence technology (TR-FRET) technology，which is used to assess the molecular-mediated interaction between the KRAS[G12C] and CypA. This method provides a high-throughput approach to detect the small molecules capable of mediating the interaction between the KRAS[G12C] and CypA simply and rapidly.
As shown in the figure below, the interaction between KRAS[G12C] and CypA 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 Daraxonrasib mediates the interaction between KRAS[G12C] and CypA, 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 Daraxonrasib, KRAS[G12C] and CypA. 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-KRAS[G12C] protein	100×	5μL	20μL	100μL	400μL	-80℃
Tag2-CypA protein	100×	5μL	20μL	100μL	400μL	-80℃
Daraxonrasib	400 μM	5μL	10μL	50μL	200μL	-80℃
Anti-Tag1 Eu antibody	50×	10μL	50μL	150μL	1000μL	-80℃
Anti-Tag2 Ac antibody	100×	5μL	25μL	125μL	500μL	-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.

操作步骤

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
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-KRAS[G12C] protein	Take 20 μL of the 100× Tag1-KRAS[G12C] 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-CypA protein	Take 20 μL of 100× Tag2-CypA protein, add it to 1.98 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 25 μL of the 100× Ac-anti-Tag2 stock solution, add 2475 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

Daraxonrasib 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. Daraxonrasib gradient dilution（Adjust according to the actual situation）

	Daraxonrasib final concentration（nM）	Daraxonrasib preparation concentration（nM）	Dilution
①	400.00	4000.00	1μL 400 μM Daraxonrasib +99μL 1× Detection buffer
②	200.00	2000.00	30μL ① +30μL 1× Detection buffer
③	100.00	1000.00	30μL ② +30μL 1× Detection buffer
④	50.00	500.00	30μL ③ +30μL 1× Detection buffer
⑤	25.00	250.00	30μL ④ +30μL 1× Detection buffer
⑥	12.50	125.00	30μL ⑤ +30μL 1× Detection buffer
⑦	6.25	62.50	30μL ⑥ +30μL 1× Detection buffer
⑧	3.13	31.25	30μL ⑦+30μL 1× Detection buffer
⑨	1.56	15.63	30μL ⑧+30μL 1× Detection buffer
⑩	0.78	7.81	30μL ⑨+30μL 1× Detection buffer
⑪	0.39	3.91	30μL ⑩+30μL 1× Detection buffer
⑫	0.20	1.95	30μL ⑪+30μL 1× Detection buffer
Blank	0	0	30μL 1× Detection buffer

2、Sample and control

2.1 Sample：Add 4 μL of Tag1-KRAS[G12C] protein working solution, 4 μL of Tag2-CypA 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 Daraxonrasib: 4 μL of Tag1-KRAS[G12C] protein working solution, 4 μL of Tag2-CypA protein working solution, 2 μL of gradient-diluted Daraxonrasib, and 10 μL of the mixed Antibody Mix sequentially into the 384-well plate.

2.3 Blank：4 μL of Tag1-KRAS[G12C] protein working solution, 4 μL of Tag2-CypA protein working solution, 2 μL of Detection buffer, and 10 μL of the mixed Antibody Mix sequentially into the 384-well plate.

2.4 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 1 hours.

Table 3. Sample and control spotting process

Daraxonrasib	Sample	Blank	Negative control (NC)
2μL gradient-diluted Daraxonrasib	2μL gradient-diluted sample	2μL 1× Detection buffer	10μL 1× Detection buffer and 10μL Antibody Mix
4μL Tag1-KRAS[G12C] protein
4μL Tag2-CypA protein
10μL Antibody Mix
Seal the plate wells with a sealing film and incubate at room temperature for 1hours.

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.