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双萤光素酶报告基因检测试剂盒

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单报告基因检测 (2) 萤光素酶 (3)
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基本描述

英文名称 Dualucif Firefly & Renilla assay kit (dual luciferase reporter 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

产品介绍：

Dualucif Firefly & Renilla Assay Kit（双萤光素酶报告基因检测试剂盒）为检测基因的表达量提供有效的手段，在 DLR检测中，萤火虫萤光素酶（Firefly luciferase）和海肾萤光素酶（Renilla luciferase）的活性可在单个样品中依次检测。先以萤光素（Luciferin）为底物来检测萤火虫萤光素酶的活性，然后加入抑制萤火虫萤光素酶催化的物质，同时加入腔肠素（Coelenterazine）检测海肾萤光素酶的活性，实现双萤光素酶报告基因检测。通过萤光素酶和其底物这一生物发光体系，可以非常灵敏、高效地检测基因的表达。通常把感兴趣基因的转录调控元件或5' 启动子区克隆在Luciferase的上游，或把3'-UTR 区克隆在Luciferase的下游，构建成报告基因（Reporter gene）质粒，然后转染细胞，用适当药物等处理细胞后裂解细胞，通过检测萤光素酶活性的高低来判断药物处理等对目的基因的转录调控作用。海肾萤光素酶更多地被用作检测转染效率的内参，以消除细胞数量和转染效率的差异。 萤火虫萤光素酶是一种分子量约为61 kDa 的蛋白，在ATP、镁离子和氧气存在的条件下，可以催化萤光素生成氧化萤光素（Oxyluciferin），在萤光素被氧化的过程中，会产生光信号。海肾萤光素酶是一种分子量约为36 kDa的蛋白，在氧气存在的条件下，可以催化腔肠素氧化成肠酰胺（Coelenteramide），在腔肠素氧化的过程中也会产生光信号。本试剂盒的光信号可以通过化学发光仪、酶标仪或液闪测定仪进行测定。该试剂盒具有检测迅速、灵敏度高、检测范围广，无细胞内源活性干扰等特点。

使用方法：
1. 细胞裂解
（1）将培养基移除，加 PBS 轻轻洗涤两次（贴壁细胞可直接进行此操作，悬浮细胞需离心收集细胞）。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（裂解产物不能反复冻融）。
（2）将裂解产物 10000-15000 rpm 离心 3-5 min，离心后将上清液移入新的 EP 管中进行后续检测。
注：细胞裂解后可立即检测，也可以冻存，需要时再检测。冻存样品需融解至室温再进行检测。
2. 工作液配制
（1）将所有组分恢复至室温。
（2）用 B 组分稀释 C 组分成 0.2 mg/mL 的萤火虫萤光素酶工作液。
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）用 D 组分将 E 组分稀释成海肾萤光素酶工作液，稀释方法为 1 µL E 组分加入到 49 µL D 组分中。
注：海肾萤光素酶工作液需现用现配。
3. 化学发光值检测
（1）按照仪器操作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）每个样品测定时，取样品 20-100 μL（如果样品量足够，请加入 100 μL；如果样品量不足可适当减少用量，但检测孔用量需保持一致）。1×Lysis Buffer 为空白对照。
（3）加入 100 μL 萤火虫萤光素酶工作液，测定 RLU（relative light unit）值（建议酶标仪设置 Shaking 混匀功能）。
注：由于该发光为瞬时发光，建议加入萤火虫萤光素酶工作液后，立即进行检测。
（4）加入 100 μL 海肾萤光素酶工作液，测定 RLU（relative light unit）值（建议酶标仪设置 Shaking 混匀功能）。
（5）在以海肾萤光素酶为内参的情况下，用萤火虫萤光素酶测定得到的 RLU 值除以海肾萤光素酶测定得到的 RLU 值。根据得到的比值来比较不同样品间目的报告基因的激活程度。如果以萤火虫萤光素酶为内参，也可以进行类似计算。

组分：


建议：

C组分建议预先使用B组分配制为2 mg/mL储液，B组分、D组分及配制为储液的C组分，根据实验需求进行小批量分装。所有检测工作液建议现配现用，避免反复冻融。

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. 为取得最佳测定效果，在用单管的化学发光仪测定时，样品和测定试剂混合后到测定前的时间应尽量控制一致；使用具有化学发光测定功能的多功能荧光酶标仪时，宜先把样品全部加好，然后统一加入萤火虫萤光素酶检测试剂。 3. 萤火虫萤光素酶催化的生物发光的最强波长为560 nm，海肾萤光素酶催化的生物发光的最强波长为480 nm。 4. 为防止孔间干扰，建议使用白色不透光孔板。 5. 由于温度对酶反应有影响，所以测定时，样品和试剂均需达到室温后再进行测定。 6. 为了您的安全和健康，请穿实验服并戴一次性手套操作。

应用范围：

基因表达调控研究、启动子研究

Product introduction：

Dualucif The firefly & Renilla assay kit (dual luciferase reporter assay kit) provides an effective means to detect the expression of genes. In DLR detection, the activities of firefly luciferase and Renilla luciferase can be detected in a single sample in turn. First, luciferin was used as substrate to detect the activity of firefly luciferase, then substances inhibiting the catalysis of firefly luciferase were added, and coelenterazine was added to detect the activity of Renilla luciferase to achieve dual luciferase reporter gene detection. The bioluminescence system of luciferase and its substrate can detect gene expression very sensitively and efficiently. Usually, the transcriptional regulatory element or 5'promoter region of the gene of interest is cloned upstream of luciferase, or the 3'-utr region is cloned downstream of luciferase to construct a reporter gene plasmid, and then transfect the cells. After the cells are treated with appropriate drugs, the cells are lysed, and the transcriptional regulation effect of drug treatment on the target gene is judged by detecting the luciferase activity. Renilla luciferase is more often used as an internal reference for detecting transfection efficiency to eliminate the difference in cell number and transfection efficiency. Firefly luciferase is a protein with a molecular weight of about 61 kDa. In the presence of ATP, magnesium ions and oxygen, it can catalyze the production of oxyluciferin from luciferin. In the process of luciferin oxidation, it will produce a light signal. Renilla luciferase is a protein with a molecular weight of about 36 kDa. In the presence of oxygen, it can catalyze the oxidation of coelenteramide to coelenteramide, and also produce light signals in the process of coelenteramide oxidation. The optical signal of this kit can be measured by chemiluminescence instrument, microplate reader or liquid scintillation tester. The kit has the characteristics of rapid detection, high sensitivity, wide detection range and no interference of cell endogenous activity.

Instruction：
1.Cell lysis
( 1 ) Remove the medium and gently wash twice with PBS ( adherent cells can be operated directly, suspension cells need to 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 were centrifuged at 10000-15000 rpm for 3-5 min. After centrifugation, the supernatant was transferred into a new EP tube for subsequent detection.
Note : Cells can be detected immediately after lysis, or frozen, and re-detected when needed. The frozen samples need to be thawed to room temperature for detection.
2. Preparation of working fluid
( 1 ) Restore all components to room temperature.
( 2 ) Dilute component C with component B to 0.2 mg / mL firefly luciferase working solution.
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 ) The E component was diluted into the renilla luciferase working solution with the D component, and the dilution method was 1 μL E component was added to the 49 μL D component.
Note : Renilla luciferase working solution needs to be prepared now.
3.chemiluminescence value detection
( 1 ) According to the operation instructions of the instrument, the instrument with chemiluminescence detection function was opened, such as multifunctional microplate reader. The parameters were set, the determination time was 10 s, and the determination interval was 2 s.
( 2 ) each sample determination, take the sample 20-100 μL ( if the sample volume is enough, please add 100 μL ; if the sample amount is insufficient, the amount can be appropriately reduced, but the amount of detection holes needs to be consistent ). 1 × Lysis Buffer was blank control.
( 3 ) 100 μL firefly luciferase working solution was added to determine the RLU ( relative light unit ) value ( it is recommended that the microplate reader set up the Shaking mixing function ).
Note : Since the luminescence is instantaneous, it is recommended to detect immediately after adding the firefly luciferase working solution.
( 4 ) 100 μL renilla luciferase working solution was added to determine the RLU ( relative light unit ) value ( Shaking mixing function is recommended for microplate reader ).
( 5 ) In the case of renilla luciferase as an internal reference, the RLU value measured by firefly luciferase was divided by the RLU value measured by renilla luciferase. According to the obtained ratio, the activation degree of the target reporter gene between different samples was compared. If firefly luciferase is used as an internal reference, similar calculations can also be performed.

Component：


Recommendation：

It is recommended to use component B in advance to prepare 2 mg / mL storage solution, component B, component D and component C prepared as storage solution, and to carry out small batch packing according to the experimental requirements. All test working fluids are recommended to be used now to avoid repeated freezing and thawing.

Matters needing attention：

1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. in order to obtain the best determination effect, when using a single tube chemiluminescence instrument for determination, the time from the mixing of sample and determination reagent to the pre determination should be controlled as much as possible; When using a multi-functional fluorescent microplate reader with chemiluminescence detection function, it is advisable to add all samples first, and then uniformly add firefly luciferase detection reagent. 3. the strongest wavelength of firefly luciferase catalyzed bioluminescence is 560 nm, and the strongest wavelength of Renilla luciferase catalyzed bioluminescence is 480 nm. 4. to prevent interference between holes, it is recommended to use white opaque orifice plate. 5. due to the influence of temperature on enzyme reaction, the sample and reagent should be measured after reaching room temperature. 6. for your safety and health, please wear experimental clothes and disposable gloves.

Scope of application：

Study on gene expression regulation and promoter

化学和物理性质

溶解性 溶于水
敏感性 对热敏感

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