产品描述

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。检测原理如图所示：

图1：萤火虫萤光素酶检测原理图

Luciferase Reporter Gene Assay kit是一闪光型萤火虫萤光素酶报告基因检测试剂盒，具有灵敏度高的特点，可以高灵敏的检测萤光素酶在哺乳动物细胞中的表达。

产品组分

运输和保存方式

干冰运输。-20℃保存，有效期1年。

试剂盒内萤火虫萤光素酶检测试剂不能反复冻融，建议分装-20℃或-80℃保存。

注意事项

1）检测过程中需自备耗材和设备包括如下：PBS；100 μL移液器或者排枪；不透光白色酶标板；Luminometer发光计、多功能酶标仪或者其他能够检测生物发光的仪器；

2）反应温度：酶促反应对温度较为敏感，加样检测前务必将所有试剂平衡至室温（20-25℃）再使用；

3）检测仪器：能检测化学发光的仪器都适用，但由于不同仪器的设置和灵敏度不同，测得的光信号值也会不同；

4）检测设置：Luminescence，350-700 nm，建议检测时间设为2-10 sec；

5）检测板：为防止孔间干扰，推荐使用不透光白色酶标板。黑色酶标板也可用，但因黑色会吸收光信号，可能会降低信号；

6）单管荧光测定仪测定，每个样品与测定试剂混合后到测定前的时间应保持一致；

7）为了您的安全和健康，请穿实验服并戴一次性手套；

8）本产品仅作科研用途！

实验步骤

I.前处理

1.细胞

1）构建相应的载体。

2）转染步骤请参照相关的说明书。

3）将细胞裂解液充分混匀，按如下方式加入细胞裂解液，充分裂解细胞。

a: 对于贴壁细胞，吸尽细胞培养液，按照下表比例加入细胞裂解液，轻轻旋转培养皿或者培养板使裂解液完全覆盖细胞；

b: 对于悬浮细胞，离心弃去上清，按照下表比例加入裂解液。

冰上孵育5 min，充分裂解细胞。

【注】：裂解产物可室温保存6 h；4℃保存16 h；-80℃可长期存放（裂解产物不能多次反复冻融），本试剂盒裂解液数量按照24孔板添加量进行配制，如需要更多可单独购买（CAT#11406）。

5）（选作）10000-16000 rpm离心1 min，取上清。

2.叶片组织（以烟草叶片为例，仅供参考）

1）构建相应的载体。

2）挑取转化有重组质粒的农杆菌单菌落，接种到2 mL LB液体培养基（添加相应抗生素）中，28℃ 220 rpm培养过夜。

3）农杆菌培养至OD600为1.0，1700× g离心5 min收集菌体后，用1/2MS液体培养基清洗菌体2次；用含有150 μmol/L乙酰丁香酮的1/2MS液体培养基将农杆菌的OD600调至1.0。

4）将待检测的农杆菌菌液进行混合，使每种菌液的OD600为0.5。

5）选取生长期为1个月左右完全伸展的烟草叶片，将混合好的菌液用1 mL注射器(去掉针头)从烟草叶背面进行注射。为保证实验结果的一致性，需要将对照载体和待检测目标载体的菌液注射在同一叶片的不同部位上, 以保证相同的生长背景。

6）正常温室生长条件下，24-48 h即可取样观察。

7）取3-4片直径为6-8 mm的叶盘，放入2 mL的 EP 管（提前放入3-4个小钢珠）中，液氮中冷冻，使用破碎仪进行研磨破碎（45 Hz，30 s）。破碎完全后在EP管中加入100 μL裂解液。

8）冰上孵育5 min左右，充分裂解叶片。

9）10000-16000 rpm离心1 min，取上清。

3.原生质体（仅供参考）

构建相应的载体。

制备原生质体（参考相关文献：Yoo SD, Cho YH, Sheen J (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2, 1565–1572）。

在2 mL EP管中加入相应的载体（加入量需要摸索），加入100 μL原生质体悬浮液。轻摇混匀后，加入110 μL PEG-CaCl2溶液，轻弹混匀。在室温放置10-15 min。

4）加入440 μL W5溶液，上下颠倒以停止转化。

5）200× g 室温离心5 min，弃去上清，加入800 μL WI溶液重悬原生质体。

室温避光培养16-24 h。

7）将原生质体加入2 mL离心管中，离心收集原生质体，加入100 μL左右的裂解液。

8）冰上孵育5 min左右，充分裂解原生质体。

9）（选做）10000-16000 rpm离心1 min，取上清。

II.荧光检测

1）按照仪器说明书要求将荧光测定仪打开，设定参数，测定时间为10 sec，测定间隔为2 sec。

2）取20 μL裂解液加入测量管中（保持每次样品的加样量一致），另外加入100 μL萤火虫萤光素酶检测试剂，震板混匀

2-3次，充分混匀后测定RLU（Relative light unit）。

3）分析数据。

①实验设计：根据不同实验目的，在每个培养板中都应设置对照组、实验组和空白对照组。为了保证实验准确性，理论上每个实验组（包括对照组）都应当减去空白对照组的萤火虫萤光素酶的发光测量值。

a.空白对照组：

背景F：未转染细胞+萤火虫萤光素酶检测试剂。

注：空白对照组的样品量必须与实验样品量相同，包含与实验样品相同的培养基/血清组合，并加上完全相同的检测试剂。

b.实验组：转染细胞经实验化合物处理(即实验组F)。

c.对照组：转染细胞不经处理，用以标准化结果(即对照组F)。

②计算结果：

实验组=实验组F-背景F。

对照组=对照组F-背景F。

表达倍数=实验组/对照组。

相关产品

 HB220325

Q：11401 有很多，现在单要裂解液？

A：有货号 11406.

Q：该试剂盒中裂解后一定要离心吗？

A：可以选做。

Q：测定时间为 10 s，测定间隔为 2 s，为什么这样推荐？如果这样设置，96 孔板这样设置，会导致每孔时间相差很大。仪器可以同时出数据。工程师建议设置动力学检测， 设置总时间。

A：不同仪器，设置时间可以不一样。单管手动检测，就按说明书设置会更好。

Q：酶标仪检测吗？

A：带有化学发光模块的可以。

Q：加入检测试剂后，产生的荧光稳定吗？多长时间内检测数据是准确的？

A：加入后海肾会有荧光猝灭的情况，越快越好，尽量 5min 内检测完成。

[1] Hao Y, Fan X, Shi Y, et al. Next-generation unnatural monosaccharides reveal that JPRRB O-GlcNAcylation regulates pluripotency of mouse embryonic stem cells. Nat Commun. 2019;10(1):4065. Published 2019 Sep 6. doi:10.1038/s41467-019-11942-y(IF:11.878)
[2] Wang X, Qin X, Yan M, et al. Loss of exosomal miR-3188 in cancer-associated fibroblasts contributes to HNC progression. J Exp Clin Cancer Res. 2019;38(1):151. Published 2019 Apr 8. doi:10.1186/s13046-019-1144-9(IF:11.161)
[3] Yin T, Liu Y, Yang M, Wang L, Zhou J, Huo M. Novel Chitosan Derivatives with Reversible Cationization and Hydrophobicization for Tumor Cytoplasm-Specific Burst Co-delivery of siRNA and Chemotherapeutics. ACS Appl Mater Interfaces. 2020;12(13):14770-14783. doi:10.1021/acsami.9b19373(IF:8.758)
[4] He Y, Qu J, Wei L, et al. Generation and Effect Testing of a SARS-CoV-2 RBD-Targeted Polyclonal Therapeutic Antibody Based on a 2-D Airway Organoid Screening System. Front Immunol. 2021;12:689065. Published 2021 Oct 18. doi:10.3389/fimmu.2021.689065(IF:7.561)
[5] Sun H, Chen D, Zhan S, et al. Design and Discovery of Natural Cyclopeptide Skeleton Based Programmed Death Ligand 1 Inhibitor as Immune Modulator for Cancer Therapy. J Med Chem. 2020;63(19):11286-11301. doi:10.1021/acs.jmedchem.0c01262(IF:6.205)
[6] Li S, Zhang L, Zhang G, et al. A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation. iScience. 2021;24(11):103296. Published 2021 Oct 16. doi:10.1016/j.isci.2021.103296(IF:5.458)
[7] Yang N, Xiong Y, Wang Y, et al. ADAP Y571 Phosphorylation Is Required to Prime STAT3 for Activation in TLR4-Stimulated Macrophages. J Immunol. 2021;206(4):814-826. doi:10.4049/jimmunol.2000569(IF:5.422)
[8] He S, Ma R, Liu Z, et al. Overexpression of BnaAGL11, a MADS-Box Transcription Factor, Regulates Leaf Morphogenesis and Senescence in Brassica napus. J Agric Food Chem. 2022;70(11):3420-3434. doi:10.1021/acs.jafc.1c07622(IF:5.279)
[9] Zhang RY, Zhou SH, He CB, et al. Adjuvant-Protein Conjugate Vaccine with Built-In TLR7 Agonist on S1 Induces Potent Immunity against SARS-CoV-2 and Variants of Concern. ACS Infect Dis. 2022;8(7):1367-1375. doi:10.1021/acsinfecdis.2c00259(IF:5.084)
[10] Li Y, Chen T, Wang W, et al. A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L. Plant Methods. 2021;17(1):106. Published 2021 Oct 16. doi:10.1186/s13007-021-00807-5(IF:4.993)
[11] Li D, Jiang K, Teng D, et al. Discovery of New Estrogen-Related Receptor α Agonists via a Combination Strategy Based on Shape Screening and Ensemble Docking. J Chem Inf Model. 2022;62(3):486-497. doi:10.1021/acs.jcim.1c00662(IF:4.956)
[12] Liu J, Chen X, Liu Y, et al. Characterization of SARS-CoV-2 worldwide transmission based on evolutionary dynamics and specific viral mutations in the spike protein. Infect Dis Poverty. 2021;10(1):112. Published 2021 Aug 21. doi:10.1186/s40249-021-00895-4(IF:4.388)
[13] Zhou FL, Li SC, Zhu Y, et al. Integrating yeast chemical genomics and mammalian cell pathway analysis [published correction appears in Acta Pharmacol Sin. 2020 May;41(5):729]. Acta Pharmacol Sin. 2019;40(9):1245-1255. doi:10.1038/s41401-019-0231-y(IF:4.010)
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产品描述

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，在氧化的过程中会发出波长为480nm左右的生物萤光。两种生物萤光都可通过化学发光仪进行测定。检测原理如图所示：

 
图1：萤火虫和海肾萤光素酶检测原理图

通常将目的基因的5´UTR或启动子克隆至Firefly Luciferase的上游，或3´UTR克隆至Firefly Luciferase的下游，通过检测萤火虫萤光素酶的量来检测启动子或调控元件的转录调控作用。Renilla Luciferase作为内参，来消除细胞数量、转染效率等的差异。Dual Luciferase Reporter Gene Assay Kit首先以萤光素为底物来检测萤火虫萤光素酶报告基因的活性，之后在淬灭该萤光反应的同时，以腔肠素为底物检测海肾萤光素酶报告基因的活性。该试剂盒具有灵敏度高的特点。

产品组分

运输和保存方式

干冰运输。 -20℃保存，有效期1年。

萤火虫萤光素酶反应工作液和海肾萤光素酶反应工作液现配现用，且不能反复冻融，建议分装-20℃或-80℃分装保存。

实验步骤

I.前处理

1.细胞

1）构建相应的载体。

2）转染步骤请参照相关的说明书。

3）将细胞裂解液充分混匀，按如下方式加入细胞裂解液，充分裂解细胞。

a: 对于贴壁细胞，吸尽细胞培养液，按照下表比例加入细胞裂解液，轻轻旋转培养皿或者培养板使裂解液完全覆盖细胞；

b: 对于悬浮细胞，离心弃去上清，按照下表比例加入裂解液。

4）冰上孵育5 min，充分裂解细胞。
【注】：裂解产物可室温保存6 h；4℃保存16 h；–80℃可长期存放。（裂解产物不能多次反复冻融）。

5）（选作）10000-16000 rpm离心1 min，取上清。

2.叶片组织（以烟草叶片为例，仅供参考）
1）构建相应的载体。
2）挑取转化有重组质粒的农杆菌单菌落，接种到2 mL LB液体培养基（添加相应抗生素）中，28℃ 220 rpm培养过夜。
3）农杆菌培养至OD₆₀₀为1.0，1700× g离心5 min收集菌体后，用1/2MS液体培养基清洗菌体2次；用含有150 μmol/L乙酰丁香酮的1/2MS液体培养基将农杆菌的OD₆₀₀调至1.0。
4）将待检测的农杆菌菌液进行混合，使每种菌液的OD₆₀₀为0.5。
5）选取生长期为1个月左右完全伸展的烟草叶片，将混合好的菌液用1 mL注射器(去掉针头)从烟草叶背面进行注射。为保证实验结果的一致性，需要将对照载体和待检测目标载体的菌液注射在同一叶片的不同部位上, 以保证相同的生长背景。
6）正常温室生长条件下，24-48 h即可取样观察。
7）取3-4片直径为6-8 mm的叶盘，放入2 mL的EP管（提前放入3-4个小钢珠）中，液氮中冷冻，使用破碎仪进行研磨破碎（45 Hz，30 s）。破碎完全后在EP管中加入100 μL裂解液。
8）冰上孵育5 min左右，充分裂解叶片。
9）10000-16000 rpm离心1 min，取上清。

3.原生质体（仅供参考）
1）构建相应的载体。
2）制备原生质体（参考文献：Yoo SD, Cho YH, Sheen J (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2, 1565–1572）。

3）WI溶液配置：0.5 M甘露醇和20mm KCl 溶于4 mm MJP (pH 5.7)，可在室温下保存。
W5溶液配置：154 mM NaCl, 125 mM CaCl₂ 和5 mM KCl溶于2 mm MJP (pH 5.7)，可在室温下保存。
4）在2 mL EP管中加入相应的载体（加入量需要摸索），加入100 μL原生质体悬浮液。轻摇混匀后，加入110 μL PEG-CaCl₂溶液，轻弹混匀。在室温放置10-15 min。
5）加入440 μL W5溶液，上下颠倒以停止转化。
6）200 × g 室温离心5 min，弃去上清，加入800 μL WI溶液重悬原生质体。
7）室温避光培养16-24 h。

8）将原生质体加入2 mL离心管中，离心收集原生质体，加入100 μL左右的裂解液。
9）冰上孵育5 min左右，充分裂解原生质体。
10）（选做）10000-16000 rpm离心1 min，取上清。

II.萤光检测

1）取20 μL裂解液，加至培养板中。按照实验需要，可设置3孔-5孔重复。

2）配制萤火虫萤光素酶反应工作液和海肾萤光素酶反应液，即萤火虫萤光素酶底物（50 ×）和海肾萤光素酶底物（50 ×）分别用对应的缓冲液稀释至1 ×工作液。并孵育至室温。

3）加入100 μL萤火虫萤光素酶反应液，震板混匀，检测萤火虫萤光素酶的活力，检测尽量在30 min内完成。

4）加入100 μL海肾萤光素酶反应液，震板混匀，检测海肾萤光素酶的活力，检测尽量在30 min内完成。

5）分析数据。

①实验设计：根据不同实验目的，在每个培养板中都应设置对照组、实验组和空白对照组。为了保证实验准确性，理论上每个实验组（包括对照组）都应当减去空白对照组的萤火虫和海肾萤光素酶的发光测量值。
 

a.空白对照组： 

背景F：未转染细胞+萤火虫萤光素酶检测试剂。

背景R：未转染细胞+萤火虫萤光素酶检测试剂+海肾萤光素酶检测试剂。

注：空白对照组的样品量必须与实验样品量相同，包含与实验样品相同的培养基/血清组合，并加上完全相同的检测试剂。

b.实验组：转染细胞经实验化合物处理(即实验组F和实验组R)。

c.对照组：转染细胞不经处理，用以标准化结果(即对照组F和对照组R)。

②计算结果：

实验组比值=(实验组F-背景F）/(实验组R-背景R)。

对照组比值=(对照组F-背景F）/(对照组R-背景R)。

表达倍数=实验组比值/对照组比值。

图2：细胞样品萤火虫和海肾萤光素酶检测流程图

注意事项

1）检测过程中需自备耗材和设备包括如下：PBS、100 μL移液器或者排枪、不透光白色酶标板、Luminometer发光计、多功能酶标仪或者其他能够检测生物发光的仪器；

2）反应温度：酶促反应对温度较为敏感，加样检测前务必将所有试剂平衡至室温（20-25℃）再使用；

3）检测仪器：能检测化学发光的仪器都适用，但由于不同仪器的设置和灵敏度不同，测得的光信号值也会不同；

4）检测设置：Luminescence，350-700 nm，建议检测时间设为2-10 sec；

5）检测板：为防止孔间干扰，推荐使用不透光白色酶标板。黑色酶标板也可用，但因黑色会吸收光信号，可能会降低信号；

6）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；

7）E组分海肾萤光素酶底物易挥发，注意密封保存；

8）为了您的安全和健康，请穿实验服并戴一次性手套。

9）本产品仅作科研用途！

相关产品

HB230504

Q：细胞裂解之后的裂解液能否在-80℃保存？

A：可保存在-80℃，基本与蛋白的保存方法类似。裂解后的样本可在 -80 ℃保存半年，-20 ℃保存一个月。

Q：双荧光素酶报告基因检测试剂盒中的两个底物是否需要避光的？

A：这两个底物操作过程中不需要严格避光。保存的时候避光保存，更重要的保存条件是低温，尤其是腔肠素，推荐–80℃保存。

Q：双荧光素酶报告基因载体共转染时比例该如何进行优化与调整？

A：比例：根据具体实验情况进行调整。建议做预实验：如海参载体与萤火虫载体比例分别用 1:10、1:20、1:50、1:100。萤火虫荧光素酶检测发光值大于海参荧光素酶发光值的比例比较好。

Q：海肾和萤火虫是在同一个孔里面检测吗？萤火虫的萤光不会影响海肾的萤光吗？能否分开检测呢？

A:同一个孔里检测。不会相互影响，我们的海肾荧光素酶底物中有淬灭萤火虫荧光值的物质。可以分开检测，但是在一个孔里检测会更加准确。

Q：使用的是promega的仪器进行检测，发现海肾的本底值偏高很多，是为什么呢？

A：我们的产品与promega的仪器不适配，会导致本底值偏高。使用酶标仪检测不会出现这种情况，建议使用酶标仪检测。

Q：W5和WI指的是什么呢？

A：

WI solution Prepare 4 mM MJP (pH 5.7) containing 0.5 M mannitol and 20 mM KCl. The prepared WI solution can be stored at room temperature (22-25 °C).
W5 solution Prepare 2 mM MJP (pH 5.7) containing 154 mM NaCl, 125 mM CaCl2 and 5 mM KCl. The prepared W5 solution can be stored at room temperature.

Q:检测萤火虫荧光素酶，酶标仪使用什么样的板子呢？

A：白色不透明的酶标板，图片见说明书

Q：裂解液不够用了，怎么办呢？

A：10ml PBS里加入0.3ml Triton

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萤火虫萤光素酶（Firefly luciferase）是一种分子量约为61kDa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，该萤光可通过化学发光仪进行测定。检测原理如图所示：

图1：萤火虫萤光素酶检测原理图

Firefly Glo Luciferase Reporter Gene Assay kit是一种辉光型萤火虫萤光素酶报告基因检测试剂盒，具有高灵敏度和发光信号稳定的特点，可以满足高通量检测萤光素酶在哺乳动物细胞中的表达。

相对于闪光型Firefly Luciferase Reporter Gene Aassy Kit（Cat#.11401），本品具有以下优点：1）发光信号更稳定性，为实验设计提供了更大的灵活性。2）采用加样-混匀-检测的操作方法，无需依赖自动进样器，并且无需弃培养液、离心等步骤，大大的简化了实验流程。3）无明显刺激性气味。

本试剂盒可用于多种常用细胞培养液：RPMI 1640、DMEM 、MEM-α、F12、DMEM/F12等，其半衰期均为2h左右（22℃），满足绝大多数高通量实验需求。

产品组分

运输与保存方式

干冰运输。未拆封试剂盒-20℃保存，有效期1年。

溶解分装后的辉光型萤火虫萤光素酶底物于-70℃避光保存1年，或-20℃短期保存不超过1个月。

使用说明

1.需自备的材料

单/多道移液器、不透光细胞培养白板、化学发光仪或带化学发光检测模块的酶标仪。

2.检测试剂准备

首次使用时将辉光型萤火虫萤光素酶缓冲液一次性全部倒入辉光型萤火虫萤光素酶底物瓶中，充分混匀后按使用需求分装，建议-70℃长期保存或-20℃保存不超过一个月。分装冻存后的检测试剂，后续每次实验前需平衡至室温。

注：辉光型萤火虫萤光素酶缓冲液可以室温或水浴融化，但温度不能超过25℃。

3.操作步骤

1）从细胞培养箱中取出含哺乳动物细胞的培养板，放置5-15 min，平衡至室温。

2）加检测试剂：加入与待测细胞培养液等体积并平衡至室温的检测试剂（例如，96孔板通常加入100 μl培养液，相应加入100 μl检测试剂；384孔板通常加入30 μl培养液，相应加入30 μl检测试剂）。

3）振荡混匀：为了使得细胞裂解充分，建议在水平摇床上室温混匀5-10 min（注：不要用移液器吹吸混匀，产生的气泡会影响发光检测读数。混匀时间可根据细胞量进行适当调整，以确保细胞充分裂解，得到稳定的发光检测结果）。

4）检测：在化学发光检测仪或带化学发光模块的多功能酶标仪上检测发光信号，加入检测试剂后2 h内完成检测。

注意事项

1）反应温度：酶促反应对温度较为敏感，加样检测前务必将检测试剂以及细胞培养液平衡至室温（20-25℃）。

2）检测仪器：能检测化学发光的仪器都适用，但由于不同仪器的设置和灵敏度不同，测得的光信号值也会不同。

3）检测板：为防止孔间干扰，推荐使用不透光细胞培养白板。

4）检测设置：Luminescence，350-700 nm，建议读板时间设为0.5-1 sec。

5）发光信号会受到检测环境如培养基组分、温度等影响，应确保同组内不同样本检测条件一致。

6）多个细胞培养板检测时，请尽量确保每板加入检测溶液后孵育时间一致，以获得最佳的检测结果。

7）为了您的安全和健康，请穿实验服并戴一次性手套。

相关产品

HB220325

Q：萤光素酶检测能用荧光显微镜吗？

A：不能。 需要带生物化学发光模块的仪器， 全波长 350-700 nm 检测。 如GloMaxTM20/20（单管检测），Biotex HTX  多功能酶标仪（配备自动进样器）。

Q：细胞裂解之后的裂解液能否在-80℃保存？

A：可保存在-80℃，基本与蛋白的保存方法类似。裂解后的样本可在 -80 ℃保存半年， -20 ℃保存一个月。

Q：荧光信号值太高该如何进行调整？

A：将裂解的样品进行稀释，或在转染实验后，减少细胞的孵育时间。

Q：应该选择单报告基因检测还是双报告基因检测？

A：

[1] Zhou Z, Zheng X, Mei X, et al. Hsa_circ_0080229 upregulates the expression of murine double minute-2 (MDM2) and promotes glioma tumorigenesis and invasion via the miR-1827 sponging mechanism. Ann Transl Med. 2021;9(9):762. doi:10.21037/atm-20-7123(IF:3.932)

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，在氧化的过程中会发出波长为480nm左右的生物萤光。两种生物萤光都可通过化学发光仪进行测定。检测原理如图所示：

图1：萤火虫和海肾萤光素酶检测原理图

Dual Glo Luciferase Reporter Gene Assay Kit是一种辉光型双萤光素酶报告基因检测试剂盒，具有高灵敏度和发光信号稳定的特点。本试剂盒中含有高纯度的萤火虫萤光素和腔肠素，在同一个样品中先以萤火虫萤光素为底物检测萤火虫萤光素酶，后淬灭萤火虫萤光素酶的萤光信号，并同时以腔肠素为底物检测海肾萤光素酶，实现双萤光素酶报告基因检测。海肾萤光素酶作为内参，消除了因孔间细胞数量、转染效率不同等造成的影响，使得检测结果的准确性更高。

相对于闪光型Dual Luciferase Reporter Gene Assay Kit（Cat NO.11402），本品具有以下优点：1）发光信号更稳定性，为实验设计提供了更大的灵活性。2）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，大大的简化了实验流程。3）无明显刺激性气味。本试剂盒可用于多种常用细胞培养液：RPMI 1640、DMEM 、MEM-α、F12、DMEM/F12等，其半衰期均为2h左右（22℃），满足绝大多数高通量实验需求。

产品组分

运输与保存方式

干冰运输。未拆封试剂盒-20℃保存，有效期1年。

溶解分装后的D-辉光型萤火虫和海肾萤光素酶底物于-70℃避光保存1年，或-20℃短期保存不超过1个月。

使用说明

1.需自备的材料

单/多道移液器；不透光细胞培养白板；化学发光仪或带化学发光检测模块的酶标仪。

2.检测试剂准备

1）萤火虫萤光素酶检测试剂：首次使用时将D-辉光型萤火虫萤光素酶缓冲液一次性全部倒入D-辉光型萤火虫萤光素酶底物瓶中，充分混匀直至底物完全溶解。按使用需求分装，建议-70℃长期保存或-20℃保存不超过一个月。

2）海肾萤光素酶检测试剂：根据实际使用量，以100:1的比例将适量的D-辉光型海肾萤光素酶缓冲液与D-辉光型海肾萤光素酶底物(100×) 混匀，室温避光备用（例如：如果需要100 mL缓冲液，则需要加入1 mL的底物），建议现配现用。

注：1）两种缓冲液都可以4℃，室温或水浴融化，但温度不能超过25℃。

2）D-辉光型海肾萤光素酶底物(100×)，每次开盖前需进行短暂低速离心。

3.操作步骤

1）从细胞培养箱中取出含哺乳动物细胞的培养板，放置5-15 min，平衡至室温。

2）萤火虫萤光素酶活性检测：

①加入与待测细胞培养液等体积并平衡至室温的萤火虫萤光素酶检测试剂（例如，96孔板建议加入80 μl培养液，相应加入80 μl检测试剂；384孔板通常加入20 μl培养液，相应加入20 μl检测试剂）。

②在水平摇床上室温混匀至少10 min。（注：不要用移液器吹吸混匀，产生的气泡会影响发光检测读数。）

③在化学发光检测仪或带化学发光模块的多功能酶标仪上检测萤火虫萤光素酶发光信号，加入检测试剂后2 h内完成检测。

3）海肾萤光素酶活性检测：

①加入平衡至室温的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充分混匀。（例如，96孔板建议加入80 μl培养液，相应加入80 μl检测试剂；384孔板通常加入20 μl培养液，相应加入20 μl检测试剂）。

②在水平摇床上室温混匀至少10 min。

③在化学发光检测仪或带化学发光模块的多功能酶标仪上检测海肾萤光素酶发光信号，加入检测试剂后2 h内完成检测。海肾萤光素酶在微孔板上的检测顺序应与萤火虫萤光素酶的检测顺序相同。

4）数据分析：

①实验设计：根据不同实验目的，在每个培养板中都应设置空白对照组，实验组和对照组。

a.空白对照组

背景F：未转染细胞+萤火虫萤光素酶检测试剂。

背景R：未转染细胞+萤火虫萤光素酶检测试剂+海肾萤光素酶检测试剂。

注：用于空白对照组样品量必须与实验样品量相同，并且包含与实验样品相同的培养基/血清组合。

b.实验组：转染细胞经实验化合物处理(即实验组F和实验组R)。

c.对照组：转染细胞不经处理，用以标准化结果(即对照组F和对照组R)。

②计算结果：实验组比值=(实验组F-背景F）/(实验组R-背景R)，对照组比值=(对照组F-背景F）/(对照组R-背景R)。

表达倍数=实验组比值/对照组比值。

注意事项

1）反应温度：酶促反应对温度较为敏感，加样检测前务必将检测试剂以及细胞培养液平衡至室温（20-25℃）。

2）检测仪器：能检测化学发光的仪器都适用，但由于不同仪器的设置和灵敏度不同，测得的光信号值也会不同。

3）检测板：为防止孔间干扰，推荐使用不透光细胞培养白板。

4）检测设置：Luminescence，350-700 nm，建议读板时间设为0.5-1 sec。

5）发光信号会受到检测环境如培养基组分、温度等影响，应确保同组内不同样本检测条件一致。

6）为了您的安全和健康，请穿实验服并戴一次性手套。

相关产品

HB220325

Q:双荧光素酶报告基因检测试剂盒中的两个底物是否需要避光的？

A:这两个底物操作过程中不需要严格避光。保存的时候避光保存，更重要的保存条件是低温， 尤其是腔肠素，推荐-80℃保存。

Q:双荧光素酶报告基因载体共转染时比例该如何进行优化与调整？

A:比例：根据具体实验情况进行调整。建议做预实验：如萤火虫载体与海参载体比例分别用 1:10、1:20、1:50、1:100。萤火虫荧光素酶检测发光值大于海参荧光素酶发光值的比例比较好

Q:萤火虫萤光素酶和海肾萤光素酶的作用及区别？

A:萤火虫萤光素酶（Firefly luciferase）：主报告基因。海肾萤光素酶（  Renilla luciferase  ）：内参报告基因。

[1] Yang D, Liu A, Zhang Y, et al. Essential Role of CRIM1 on Endometrial Receptivity in Goat. Int J Mol Sci. 2021;22(10):5323. Published 2021 May 18. doi:10.3390/ijms22105323(IF:5.924)
[2] Shen L, Ji C, Lin J, Yang H. Regulation of circADAMTS6-miR-324-5p-PIK3R3 ceRNA pathway may be a novel mechanism of IL-1β-induced osteoarthritic chondrocytes. J Bone Miner Metab. 2022;40(3):389-401. doi:10.1007/s00774-021-01308-0(IF:2.626)
[3] Chen B, Wang Y, Pei X, Wang S, Zhang H, Peng Y. Cellular Caspase-3 Contributes to EV-A71 2A^pro-Mediated Down-Regulation of IFNAR1 at the Translation Level. Virol Sin. 2020;35(1):64-72. doi:10.1007/s12250-019-00151-y(IF:2.467)
[4] Yang J, Liu B, Xu Z, Feng M. Silencing of Circ_0135889 Restrains Proliferation and Tumorigenicity of Human Neuroblastoma Cells [published online ahead of print, 2022 Jun 27]. J Surg Res. 2022;279:135-147. doi:10.1016/j.jss.2022.05.025(IF:2.192)