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Bio-UltraBio™ 双萤光素酶一步检测试剂盒
有货
为什么选择此级别
BioReagent,用于化学发光 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
-80℃储存。特低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
报告基因已被细胞生物学广泛使用以研究基因表达及其它与基因表达相关的细胞学事件，如受体活性、细胞内信号传导、mRNA 加工、蛋白质折叠及蛋白-蛋白相互作用等。萤火虫萤光素酶和海肾萤光素酶是基因表达中最常用的两种报告基因，是极度灵敏、响应快速且易于使用的报告基因，所催化的化学发光反应是测量基因表达最敏感的分析工具之一。
萤火虫萤光素酶（Firefly luciferase）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，能够催化萤光素（luciferin）氧化成oxyluciferin，在luciferin氧化的过程中会发出波长为560 nm左右的生物萤光；海肾萤光素酶（Renilla luciferase）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（Coelenterazine）氧化成Coelenteramide。在Coelenterazine氧化的过程中，会发出波长为480nm左右的生物萤光，这两种萤光都可以通过化学发光仪进行测定。
Bio-UltraBio™ 双萤光素酶一步检测试剂盒是一种辉光型检测试剂盒，具有高灵敏度和发光信号稳定的特点，符合高通量检测的需求。该检测试剂盒在同一个样品中先以萤光素为底物来检测萤火虫萤光素酶，后以腔肠素为底物来检测海肾萤光素酶，同时淬灭萤火虫萤光素酶的萤光信号，实现双萤光素酶报告基因检测。以海肾萤光素酶为内参，可校正孔间细胞铺板量、转染效率不均一带来的检测偏差，有效提升数据准确度。
相比于Bio-UltraBio™ 双萤光素酶分步检测试剂盒（D1375165），本产品具有以下优点：两种萤光素酶的半衰期约1小时，其中萤火虫萤光素酶最长可达2小时，发光信号稳定持久，为实验设计提供了更大的灵活性；更符合高通量检测，无需依赖自动进样器，并且无需弃培养液、离心等步骤，简化了实验流程。
注意事项：
1、 酶促反应对温度较为敏感，加样检测前需将细胞培养板和检测液平衡至室温。
2、 检测仪器选择：能够检测化学发光的仪器都适用本试剂盒的检测，但是针对相同的样品，不同检测器本底信号值和测量值均可能不同；且对于同一样本检测，不同仪器的数值不可横向比较。
3、 为防止孔间干扰，推荐使用不透明白色或黑色细胞培养板。
4、 海肾萤光素酶检测底物（100×）配制在无水乙醇中。由于无水乙醇容易挥发，有时会在初次使用时发现体积明显小于包装规格的情况，此时用无水乙醇把体积补足至包装规格，并混匀后即可使用。
5、 当酶表达量过低时，发光信号会降低，但信号稳定性会增强；当酶表达量过高时，信号半衰期会减少，但发光信号会增强。建议根据实际情况优化实验设计方案（如增加/减少质粒转染量），避免萤光素酶表达量过高/低。
使用说明：
1. 试剂准备
（1）海肾萤光素酶检测底物（100×）：每次开盖前需进行短暂低速离心
（2）海肾萤光素酶检测试剂配制：根据实际使用量，以 99:1 的比例将适量的海肾萤光素酶检测缓冲液与海肾萤光素酶检测底物（100×）混匀，避光备用。
注：建议现配现用，剩余的检测试剂当次实验结束后，直接舍弃，不要留存。
2. 萤火虫萤光素酶检测
（1）加入萤火虫萤光素酶检测试剂，加样体积与细胞培养液体积相同并混匀。由于需要分别加入两种检测试剂，为防止孔板液体溢出，96孔板建议加入80μl培养液，相应加入80μl萤火虫萤光素酶检测试剂。
（2）室温孵育5min。
（3）孵育后于化学发光仪或酶标仪中检测萤火虫萤光素酶报告基因活性。
3. 海肾萤光素酶检测
（1）加入海肾萤光素酶检测试剂，加样体积与初始细胞培养液体积相同并混匀。例如，96孔板建议加入80μl培养液，相应加入80μl海肾萤光素酶检测试剂。
（2）室温孵育5min。
（3）孵育后于化学发光仪或酶标仪中检测海肾萤光素酶报告基因活性。
Genetic reporters are used commonly in cell biology to study gene expression and other cellular events coupled to gene expression, such as receptor activity, intracellular signal transduction, mRNA processing, protein folding and protein:protein interactions. Firefly luciferase and Renilla luciferase are the two most commonly used reporter genes in gene expression studies. These reporters are highly sensitive, respond rapidly, and are easy to use, with their catalyzed chemiluminescent reactions serving as one of the most sensitive analytical tools for measuring gene expression.
Firefly luciferase is a protein with a molecular weight of approximately 61 kDa that catalyzes the oxidation of luciferin to oxyluciferin in the presence of ATP, magnesium ions, and oxygen. This oxidation process produces bioluminescence with a peak wavelength around 560 nm. Renilla luciferase, a protein with a molecular weight of about 36 kDa, catalyzes the oxidation of coelenterazine to coelenteramide in the presence of oxygen, generating bioluminescence with a peak wavelength around 480 nm. Both luminescent signals can be detected using a chemiluminescence instrument.
The Bio-UltraBio™ Dual Luciferase One-Step Reporter Gene Assay Kit is a glow-type detection kit characterized by high sensitivity and stable luminescent signal, making it well-suited for high-throughput screening. This kit first detects firefly luciferase activity using luciferin as substrate, then detects Renilla luciferase activity using coelenterazine as substrate while simultaneously quenching the firefly luciferase signal, enabling simultaneous dual luciferase reporter gene analysis. By using Renilla luciferase as an internal control, variations in cell seeding density and transfection efficiency across wells can be corrected, significantly improving data accuracy.
Compared to the Bio-UltraBio™ Dual Luciferase Two-Step Reporter Gene Assay Kit (D1375165), this product offers several advantages: both luciferases have half-lives of approximately one hour, with firefly luciferase lasting up to two hours, providing stable and long-lasting luminescent signals that offer greater flexibility in experimental design; it is better suited for high-throughput applications, requiring no automated liquid handling system, and eliminates steps such as removing culture medium or centrifugation, thereby simplifying the experimental workflow.
Precautions
1. The enzymatic reaction is temperature-sensitive. Both the cell culture plate and the detection reagent must be equilibrated to room temperature prior to assay initiation.
2. Detector Selection: Any instrument capable of measuring chemiluminescence is suitable for use with this kit. However, for identical samples, the background signal and measured values may vary between different detectors. Furthermore, readings obtained from different instruments for the same sample are not directly comparable.
3. To prevent well-to-well crosstalk, the use of opaque white or black cell culture plates is recommended.
4. Renilla luciferase substrate (100×) is prepared in anhydrous ethanol. Since anhydrous ethanol is prone to volatilization, sometimes it is observed that the volume is significantly smaller than the packaging specification during the initial use. In such cases, the volume can be replenished to the packaging specification using anhydrous ethanol, and then mixed well before use.
5. When enzyme expression is too low, the luminescent signal decreases but stability increases; when enzyme expression is too high, the signal half-life shortens but the luminescent signal intensifies. It is recommended to optimize the experimental design according to actual conditions (e.g., increasing or decreasing plasmid transfection amount) to avoid excessively high or low luciferase expression.
Instruction for use
1. Reagent preparation
(1) Renilla luciferase substrate (100×): Before each opening of the container, a brief low-speed centrifugation is required.
(2) Preparation of renilla luciferase detection reagents: Based on the actual usage amount, mix an appropriate amount of Renilla luciferase detection buffer with the Renilla luciferase substrate (100×) in a 99:1 ratio. Store in the dark for later use.
Note: It is recommended to prepare the reagents immediately before use. The remaining detection reagents should be discarded at the end of the current experiment and not retained.
2. Firefly Luciferase Assay
(1) Add firefly luciferase detection reagent, using the same volume as the cell culture medium and mix thoroughly. Since two detection reagents need to be added separately, to prevent liquid overflow in the plate, it is recommended to add 80 μL of culture medium into a 96-well plate, along with 80 μL of firefly luciferase detection reagent.
(2) Incubate at room temperature for 5 minutes.
(3) After incubation, measure the activity of the firefly luciferase reporter gene using a chemiluminescence reader or microplate reader.
3. Renilla Luciferase Assay
(1) Add renilla luciferase detection reagent, using the same volume as the initial cell culture medium and mix thoroughly. For example, in a 96-well plate, it is recommended to add 80 μL of culture medium and 80 μL of renilla luciferase detection reagent accordingly.
(2) Incubate at room temperature for 5 minutes.
(3) After incubation, measure the activity of the renilla luciferase reporter gene using a chemiluminescence reader or microplate reader.
规格
别名
双萤光素酶报告基因检测试剂盒 | 辉光型双萤光素酶报告基因检测试剂盒
英文别名
Firefly&Renilla-Light Luciferase Reporter Assay Kit | Dual Luciferase Reporter Gene Assay Kit
规格或纯度
BioReagent, 用于化学发光
英文名称
Bio-UltraBio™ Dual Luciferase One-Step Reporter Gene Assay Kit
应用
Chemiluminescence
样品类型
cell or cell lysate.
检测方法
Chemiluminescence
特异性
Firefly luciferase; Renilla luciferase
检测仪器
化学发光仪,multiscan spectrum
敏感性
light&Temperature&Freeze-thaw sensitive
储存与运输
储存条件
-80℃储存
运输条件
特低温运输
稳定性与储存
各组分在相应的储存条件下保质期1年。
组分与储存
D1375164 | Components | Appearance | 100T | 1000T | 储存 | 单次测试使用量 |
D1375164A | 萤火虫萤光素酶检测试剂 | 液体 | 10 mL | 100mL | -80℃，避光 | 80 μL |
D1375164B | 海肾萤光素酶检测缓冲液 | 液体 | 10 mL | 100mL | -80℃，避光 | 79 μL |
D1375164C | 海肾萤光素酶检测底物（100×） | 液体 | 0.1 mL | 1 mL | -80℃，避光 | 1 μL |
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