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Firefly Luciferase Assay Kit（萤火虫萤光素酶报告基因检测试剂盒）

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货号 (SKU) 包装规格 是否现货 价格 数量
F598310-50T
 50T 期货 Stock Image
F598310-200T
 200T 期货 Stock Image
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报告基因检测 (28) 生物发光 (27)
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基本描述

英文名称 Firefly luciferase assay kit
储存温度 -20°C储存
运输条件 超低温冰袋运输
产品介绍

真核基因表达调控研究常用的方法是进行报告基因的检测，生物发光法又是报告基因检测最常用的有效手段。萤光素酶能催化底物萤光素的转化，并发射出光子。该产品为萤火虫萤光素酶报告基因在哺乳动物细胞中的表达提供快速、灵敏、稳定的检测方法。能够检测最低10-20 mol的萤光素酶，在10-14至10-20mol的酶浓度范围内呈很好的线性关系。

组分：


使用方法：
1. 细胞裂解
（1）将转入报告基因的细胞中的培养基移除，加 PBS 轻轻洗涤（贴壁细胞可直接进行此操作，悬浮细胞要离心收集细胞）。按如下方案加入 1×Lysis Buffer(用无菌水按 4:1 稀释 A 组分)，然后将培养板放在微型震荡器上室温震荡 15 min，充分裂解细胞。


注：裂解产物可室温保存 6 h，-70℃可长期存放（裂解产物不能多次反复冻融）。
（2）将充分裂解后的裂解产物，10000-15000 rpm 离心 3-5 min，收上清。
2. 工作液配置
（1）将所有组分恢复至室温。
（2）用 B 组分充分稀释 C 组分（储液），配制成 0.2 mg/mL 的萤火虫萤光素酶工作液，涡旋震荡，确保充分混匀。
注：萤火虫萤光素酶工作液不能反复冻融，若单次实验用量较少，建议按单次使用量分装成小规格。
3. 化学发光值检测
（1）按仪器说明书开启具有检测化学发光功能的仪器，如多功能酶标仪。设定参数，测定时间为 10 s，测定间隔为 2 s。
（2）将细胞裂解产物按照 20~100 µL 的体积加入测量管中（保持每次样品量一致）。1× Lysis Buffer 为空白对照。
（3）加入 100 µL 萤火虫萤光素酶检测液，测定 RLU（Relative light unit）（建议酶标仪设置 Shaking 混匀功能）。

注意事项：

1. 使用前请将产品瞬时离心至管底，再进行后续实验。

2. 温度会对酶的活性产生影响，因此所有试剂应放置室温后再使用。

3. 如果单管萤光测定仪测定，每个样品与测定试剂混合后到测定前的时间应保持一致。

4. 萤火虫萤光素酶催化的生物发光的最强波长为560 nm。

5. 为防止孔间干扰，建议使用白色不透光孔板。

6. 为了您的安全和健康，请穿实验服并戴一次性手套操作。

产品特点：

1. 快速： 快速：细胞裂解在 10-15 min 内完成；

2. 方便：试剂易于配制，样品检测步骤简单；

3.灵敏：能够检测最低 10-20 mol 的萤光素酶；

4. 酶的浓度线性范围可达 8 个数量级。

建议：

C 组分建议预先使用无菌水配置为 2 mg/mL 储液，B 组分及配置为储液的 C 组分，根据实验需求进行小批量分装。检测工作液建议现配现用，避免反复冻融。

应用范围：

基因表达调控研究、启动子研究

The commonly used method of eukarYOtic gene expression regulation research is the detection of reporter genes, and bioluminescence is the most commonly used and effective means of reporter gene detection. Luciferase can catalyze the conversion of the substrate luciferin and emit photons. This product provides a rapid, sensitive and stable detection method for the expression of firefly luciferase reporter gene in mammalian cells. It can detect the lowest luciferase of 10-20 mol, and has a good linear relationship in the concentration range of 10-14 to 10-20mol.

Component：


Instruction：
1. Cell lysis
( 1 ) The medium in the cells transfected with the reporter gene was removed and gently washed with PBS ( adherent cells can be directly subjected to this operation, and suspended cells should be centrifuged to collect cells ). Add 1 × Lysis Buffer ( diluted component A with sterile water at 4 : 1 ) according to the following scheme, and then place the culture plate on a micro-oscillator at room temperature for 15 min to fully lyse the cells. 


Note : The pyrolysis products can be stored at room temperature for 6 h, and can be stored at − 70 °C for a long time ( the pyrolysis products cannot be repeatedly frozen and thawed ).
( 2 ) The pyrolysis products after full pyrolysis were centrifuged at 10000-15000 rpm for 3-5 min, and the supernatant was collected.
2.Working fluid configuration
( 1 ) Restore all components to room temperature.
( 2 ) The component C ( storage solution ) was fully diluted with component B to prepare a 0.2 mg / mL firefly luciferase working solution, which was vortexed and shaken to ensure full mixing.
Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the amount of a single experiment is small, it is recommended to be subpackaged into small specifications according to a single amount of use.
3.chemiluminescence value detection
( 1 ) According to the instructions of the instrument, open the instrument with chemiluminescence detection function, such as multifunctional microplate reader. The parameters were set, the determination time was 10 s, and the determination interval was 2 s.
( 2 ) The cell lysis products were added to the measuring tube according to the volume of 20 ~ 100 μL ( keep the same amount of samples each time ). 1 × Lysis Buffer was blank control.
( 3 ) 100 μL firefly luciferase detection solution was added to determine RLU ( Relative light unit ) ( Shaking mixing function is recommended for microplate reader ).

Matters needing attention：

1.Please instantaneously centrifuge the product to the bottom of the tube before use, and then carry out subsequent experiments. 

2.Temperature will affect the activity of the enzyme, so all reagents should be placed at room temperature before use. 

3.If the single-tube fluorescence detector is used, the time from the mixing of each sample with the determination reagent to the time before the determination should be consistent. 

4.The strongest wavelength of bioluminescence catalyzed by firefly luciferase is 560 nm. 

5.In order to prevent interference between holes, it is recommended to use a white opaque hole plate. 

6.For your safety and health, please wear experimental clothes and wear disposable gloves.

Product characteristic：

1.Rapid : Rapid : Cell lysis was completed within 10-15 min ; 

2.Convenience : the reagent is easy to prepare, the sample detection steps are simple ; 

3.Sensitive : capable of detecting a minimum of 10-20 mol luciferase ; 

4.the linear range of enzyme concentration can reach 8 orders of magnitude.

Recommendation：

Component C is recommended to use sterile water in advance to prepare 2 mg / mL stock solution.B component and C component configured as storage solution, and small batch packing according to the experimental requirements. The detection working fluid is recommended to be used now to avoid repeated freezing and thawing.
Scope of application：

Study on gene expression regulation and promoter

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批号(Lot Number) 证书类型 货号
C2518622 分析证书 F598310

溶液计算器

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