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Bio-UltraBio™高亮版萤火虫萤光素酶分步检测试剂盒（冻干型）
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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
避光,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
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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。萤火虫荧光素酶是基因表达中最常用的报告基因之一，是一种极度灵敏、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左右的生物荧光，该萤光可通过化学发光仪进行测定。
Bio-UltraBio™高亮版萤火虫萤光素酶分步检测试剂盒（冻干型）是一种高灵敏度辉光型定量检测试剂盒，特别适用于萤火虫荧光素酶低表达的样品检测。该试剂盒是[B1373068]Bio-UltraBio™高亮版萤火虫萤光素酶分步检测试剂盒（即用型）的不同包装版本，两者的检测信号强度相当，信号半衰期均约30分钟。[B1373068]Bio-UltraBio™高亮版萤火虫萤光素酶分步检测试剂盒（即用型）的检测液为即用型液体，其优点是无需配制即可直接使用，但需要-80℃保存，如果在-20℃保存时间较长后检测效果会逐渐下降。本产品为冻干粉版本，在-20℃保存非常稳定，初次使用时需用提供的检测缓冲液溶解检测底物。试剂盒提供的检测缓冲液可存放于25℃以下室温环境，节约了试剂的平衡时间。
注意事项：
1、 5×报告基因细胞裂解液可在4℃短期存放，-20℃保存一年有效，-80℃可长期存放。
2、 初次使用时，需用提供的萤火虫萤光素酶检测缓冲液复溶萤火虫萤光素酶底物。
3、 复溶后的萤火虫荧光素酶底物检测液应尽量避免多次反复冻融。建议第一次复溶后适当分装，然后避光保存于-20℃或-70℃。
4、 复溶后的萤火虫荧光素酶底物检测液避免长时间暴露于室温。
5、 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。
6、 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。
7、 为防止孔间干扰，推荐使用不透明白色或黑色细胞培养板。
使用说明：
1. 细胞裂解
1) 贴壁细胞：吸尽培养液。
悬浮细胞：离心收集细胞，弃上清。
2) 根据培养器皿类型，参考下表加入适量1×Lysis Buffer (用无菌水按 4:1 稀释 A 组分)：
器皿类型 | 96孔板 | 48孔板 | 24孔板 | 12孔板 | 6孔板 |
裂解液体积 (μl/孔) | 100 | 150 | 200 | 300 | 500 |
*注：若萤光素酶表达水平较低，可酌情减少裂解液用量（例如，6孔板可低至100μl/孔）。
3) 室温条件下，在微型震荡器上或静置裂解10min。
4）96孔板样品可直接进行后续检测，也可以先冻存，待以后再测定。冻存样品需融解，并达到室温后再进行测定。
5）其他类型样品10,000-15,000×g离心3-5分钟，取上清用于测定。
2. 试剂准备
将萤火虫萤光素酶检测缓冲液全部倒入萤火虫萤光素酶底物，上下颠倒瓶身数次，充分溶解底物。
【注】复溶后的萤火虫萤光素酶检测液一次实验使用不完时，建议复溶后直接在冰上分装，把多余的检测试剂冻存于-20℃或-80℃。
3. 仪器设置
启动化学发光检测仪或具备化学发光功能的多功能酶标仪，根据仪器说明书及实验需求进行参数设置。
4. 加样
取20-100μl细胞裂解样品进行检测（建议用量100μl；若样本量有限可减少用量，但同批次实验应保持一致）。同时，设置等体积裂解液作为空白对照。
5. 检测
向各孔中加入100μl复溶后的萤火虫萤光素酶底物检测液，立即上机读取信号值。
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 is one of the most commonly used reporter genes in gene expression. It is an extremely sensitive, fast-responding and easy-to-use reporter gene. The chemiluminescence reaction it catalyzes is one of the most sensitive analytical tools for measuring gene expression.
Firefly luciferase is a protein with a molecular weight of approximately 61 kDa. In the presence of ATP, magnesium ions and oxygen, it can catalyze the oxidation of luciferin into oxyluciferin. During the oxidation process of luciferin, bioluminescence with a wavelength of approximately 560 nm is emitted, and this fluorescence can be measured by a chemiluminescence analyzer.
Bio-UltraBio ™ High brightness Firefly Fluorescent Enzyme Step by Step Detection Kit (Freeze Dried Type) is a highly sensitive, glow-type quantitative detection kit, particularly suitable for samples with low expression levels of firefly luciferase. This kit is an alternative packaging version of the [B1373068Z] Bio-UltraBio™ Bright two-step Firefly Luciferase Reporter Gene Assay Kit (Ready-to-use）, with comparable detection signal intensity. Both kits exhibit a signal half-life of approximately 30 minutes.
The detection reagent in the [B1373068Z] Bio-UltraBio™ Bright two-step Firefly Luciferase Reporter Gene Assay Kit (Ready-to-use） is supplied as a ready-to-use liquid, offering the advantage of direct use without reconstitution. However, it requires storage at -80°C, and prolonged storage at -20°C may gradually reduce its detection performance.
In contrast, this lyophilized powder product remains highly stable when stored at -20°C. For initial use, the firefly luciferase substrate must be reconstituted with the provided firefly luciferase detection buffer. The assay buffer included in the kit can be stored at room temperature (≤ 25°C), thereby saving time required for temperature equilibration.
Precautions
1. The 5× Luciferase Assay Lysis Buffer can be stored short-term at 4°C, is stable for up to one year at -20°C, and can be stored long-term at -80°C.
2. For initial use, the firefly luciferase substrate must be reconstituted with the provided Firefly Luciferase Assay Buffer.
3. Repeated freeze-thaw cycles of the reconstituted Firefly Luciferase Substrate Detection Reagent should be minimized. It is recommended to aliquot the solution upon initial reconstitution and store it protected from light at -20°C or -80°C.
4. Avoid prolonged exposure of the reconstituted Firefly Luciferase Substrate Detection Reagent to room temperature.
5. 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.
6. 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.
7. To prevent well-to-well crosstalk, the use of opaque white or black cell culture plates is recommended.
Instruction for use
1. Cell lysis
1) Adherent cells: Aspirate the culture medium completely.
Suspension cells: Centrifuge to collect the cells and discard the supernatant.
2) Add an appropriate volume of 1× Lysis Buffer (prepared by diluting Component A 4:1 with sterile water) according to the culture vessel, as referenced in the table below:
Culture Vessel | 96-well plate | 48-well plate | 24-well plate | 12-well plate | 6-well plate |
Lysis Buffer Volume (μl/well) | 100 | 150 | 200 | 300 | 500 |
*Note: If luciferase expression level is low, consider using less lysis buffer (e.g., as low as 100μl/well for a 6-well plate).*
3) Lyse the cells for 10 minutes at room temperature, either on a microplate shaker or by standing.
4) For 96-well plates: Lysates can be assayed directly or frozen for later analysis. Thaw frozen samples completely and equilibrate to room temperature before assaying.
5) For other vessel types: Centrifuge lysates at 10,000-15,000 × g for 3-5 minutes. Use the supernatant for the assay.
2. Reagent preparation
Completely pour the Firefly Luciferase Detection Buffer into the vial containing the Firefly Luciferase Substrate. Invert the vial several times to fully dissolve the substrate.
Note: If the reconstituted Firefly Luciferase Detection Reagent is not fully used in a single experiment, it is recommended to aliquot it on ice immediately after reconstitution. The remaining aliquots should be stored at -20°C or -80°C.
3. Instrument Settings
Turn on the luminometer or a multifunctional microplate reader with chemiluminescence detection capability. Set instrument parameters according to the manufacturer's instructions and experimental requirements.
4. Sample Loading
Load 20-100 μl of cell lysate (or supernatant for centrifuged samples) for measurement (100 μl is recommended if sample volume permits; use a consistent volume across all samples in the same experiment). Use an equal volume of lysis buffer as a blank control.
5. Measurement
Add 100 μl of the Firefly Luciferase Detection Reagent to each sample. Mix thoroughly and measure the luminescent signal immediately.
规格
别名
萤火虫萤光素酶报告基因检测试剂盒II (增强型) | 萤火虫萤光素酶报告基因检测试剂盒II (增强型) | 萤火虫萤光素酶报告基因检测试剂盒 | 萤光素酶检测系统
英文别名
Single-Luciferase (Firefly) Reporter Assay Kit | Firefly Luciferase Assay Kit；Luciferase Assay System | Single-Luciferase (Firefly) Reporter Assay Kit | Firefly Luciferase Assay Kit | Luciferase Assay System
规格或纯度
BioReagent, 用于化学发光
英文名称
Bio-UltraBio ™ High brightness Firefly Fluorescent Enzyme Step by Step Detection Kit (Freeze Dried Type)
应用
Chemiluminescence
样品类型
cell or cell lysate
检测方法
Chemiluminescence
特异性
Firefly luciferase
检测仪器
酶标仪,化学发光仪
检测波长
560
敏感性
light-sensitive；Temperature-sensitive；Freeze-thaw sensitive
储存与运输
储存条件
避光,-20°C储存
运输条件
超低温运输
稳定性与储存
Bio-UltraBio™高亮版萤火虫萤光素酶分步检测试剂盒（冻干型）：各组分在相应的储存条件下保质期1年。
组分与储存
B1506973 | Components | 100T | 10 × 100T | 储存条件 | 用量 / 次 |
B1506973A | 5×报告基因细胞裂解液 | 10 mL | 10×10ml | -20℃ | 100μL |
B1506973B | 萤火虫荧光素酶检测缓冲液 | 10 mL | 10×10ml | -20℃ | 100μL |
B1506973C | 萤火虫荧光素酶检测底物 | 1瓶 | 10瓶 | -20℃, 避光保存 | 100μL（复溶后） |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0333652 |
分析证书 | B1506973 |
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