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双萤光素酶报告基因检测试剂盒Dual Luciferase Reporter Gene Assay Kit

双萤光素酶报告基因检测试剂盒(Dual Luciferase Reporter Gene Assay Kit)，是先以萤光素(luciferin)为底物来检测萤火虫萤光素酶(Firefly luciferase)，后以肠腔素(coelenterazine)为底物来检测海肾萤光素酶(Renilla luciferase)，并且在后续加入海肾萤光素酶底物时，同时加入抑制萤火虫荧光素酶催化luciferin发光的物质，使后续检测仅仅检测到海肾萤光素酶的活性，实现双萤光素酶报告基因检测。萤火虫萤光素酶是一种分子量约为61kD的蛋白，在ATP、镁离子和氧气存在的条件下，可以催化luciferin氧化成oxyluciferin，在luciferin氧化的过程中，会发出生物萤光(bioluminescence)。海肾萤光素酶是一种分子量约为36kD的蛋白，在氧气存在的条件下，可以催化coelenterazine氧化成coelenteramide，在coelenterazine氧化的过程中也会发出生物萤光。生物萤光可以通过化学发光仪(luminometer)或液闪测定仪进行测定。

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1000

产品编号

RG088-100T/680元

所属分类

BioAssay Kits

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

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双萤光素酶报告基因检测试剂盒

Dual Luciferase Reporter Gene Assay Kit

Introduction：

Firefly luciferase is widely used as a reporter for studying gene regulation and function, and for pharmaceutical screening. It is a very sensitive genetic reporter due to the absence of endogenous luciferase activity in mammalian cells or tissues. Firefly luciferase, a monomeric 62,000 Dalton protein, catalyzes ATP-dependent D-luciferin oxidation to oxyluciferin to produce light at 560nm.

Renilla luciferase has been used as a reporter gene for studying gene regulation and function in vitro and in vivo. It commonly is used in multiplex transcriptional reporter assays or as a normalizing transfection control for firefly luciferase assays. Renilla luciferase, a monomeric 36,000 Dalton protein, catalyzes coelenterazine oxidation by oxygen to produce light at 465nm.

The accuracy of reporter assays can be improved by utilizing a dual reporter system. One of the reporter genes is correlated with the promoter of interest and is used to assess the effects of specific experimental conditions, while the second reporter is used as a control and serves as a baseline response. Our Dual Luciferase Reporter Gene Assay Kit allows for the sequential measurement of the activity of two different luciferases, firefly (FFL) and Renilla (RL), in a single sample. The firefly luciferase luminescence is measured first by addition of the FFL Reagent. Next, the RL Reagent is added to the same well. The RL Reagent simultaneously quenches the firefly luciferase luminescence and initiates the Renilla luciferase reaction. The light production of both reactions can be conveniently measured on a luminometer.

This bioluminescent dual reporter gene assay is extremely sensitive and is especially suitable for quantifying dual luciferase expression in recombinant cells or in cell free transcription/translation reactions. Assays can be performed in tubes, cuvettes or multi-well plates.

截屏2022-10-08 13.15.15.png

Figure 1. Bioluminescent reactions catalyzed by firefly luciferase and Renilla luciferase.

Components of the kit：

Cat No	Product Name	Size	Price
RG088	Dual Luciferase Reporter Gene Assay Kit	100T	680RMB
Cat No	Components	Quantity	Storage
RG088-A	1X Passive Luciferase Lysis Buffer	10ml	-20°C Protect from light 1 year
RG088-B	Firefly Luciferase Assay Buffer	10ml	-20°C Protect from light 1 year
RG088-C	D-Luciferin	2mg	-80°C Protect from light 1 year
RG088-D	Renilla Luciferase Assay Buffer	10mL	-20°C Protect from light 1 year
RG088-E	Coelenterazine	400ug	-80°C Protect from light 1 year

Storage and Handling:

Store the kit at -80°C. Firefly and Renilla Assay Buffers are stable at -80°C for at least six months from date of receipt. Other components are stable at -20°C or below for at least six months from date of receipt. Kit components and stock solutions of D-Luciferin and Aquaphile Coelenterazine in water are stable to at least 5 freeze/thaw cycles.

Protocol：

1. Preparation of cell lysates

Note: 1X Passive Lysis Buffer is ready to use without dilution.

1.1 Remove the growth medium from the cultured cells and gently wash the cells once with a sufficient volume of phosphate buffered saline (PBS) to cover the surface of the culture vessel. Remove the PBS and add 1X Passive Lysis Buffer 2.0 using the volume recommended below for each type of well:

Cell Culture Plate	96-well plates	48-well plates	24-well plates	12-well plates	6-well plates
Lysis Buffer (A) uL/per well	20uL	65uL	100uL	250uL	500uL

1.2 Place the culture plates on a rocking platform or orbital shaker with gentle rocking/shaking to ensure complete and even coverage of the cell monolayer with 1X passive lysis buffer. Rock the culture plates at room temperature for 15 minutes.

Note: Cultures that are overgrown are often more resistant to complete lysis and typically require an increased volume of passive lysis buffer and/or an extended treatment period to ensure complete lysis and/or scraping cells off the culture plates.

Note: 1X Passive Lysis Buffer contains protein stabilizers that may affect results of protein quantitation assays.

1.3 Transfer the lysate to a tube or vial. Optional: the lysate can be cleared by centrifugation for 30 seconds at top speed in a refrigerated microcentrifuge and transferred into a new tube. Place at 4°C until ready to assay. Lysates can be stored at -20°C or -80°C for up to two weeks.

2. Preparation of Firefly Working Solution

2.1 Thaw Firefly Luciferase Assay Buffer 2.0 at room temperature.

2.2 Prepare 10 mg/mL D-luciferin stock solution. For component C (2mg), add 200 uL water to the vial and mix. The stock solution can be stored for at least 6 months at -20°C or below, and is stable to up to 5 freeze/thaw cycles.

2.3 Prepare enough firefly working solution to perform the desired number of assays (100 uL working solution per assay). Dilute D-luciferin (10 mg/mL) in assay buffer at a ratio of 1:50. For example, add 20 uL D-luciferin stock solution to 1 mL firefly assay buffer.

Note: For best results, working solutions (assay buffer with substrate) should be prepared fresh before each use, and used within 3 hours of preparation. Firefly working solution activity decreases ~10% after 3 hours and ~25% after 5 hours at room temperature.

3. Preparation of Renilla Working Solution

1.1 Thaw Renilla Luciferase Assay Buffer at room temperature.

1.2 Prepare 2 mg/mLcoelenterazine stock solution. For component E-400ug, add 200 uL water to the vial and mix. The stock solution can be stored for up to 3 months at -20°C or below, and is stable to up to 5 freeze/thaw cycles.

1.3 Prepare enough Renilla working solution to perform the desired number of assays (100 uL working solution per assay). Dilute coelenterazine (2 mg/mL) in Renilla Luciferase Assay Buffer at a ratio of 1:50. For example, add 20 uL coelenterazine stock solution to 1 mL assay buffer.

Note: For best results, working solutions (assay buffer with substrate) should be prepared fresh before each use, and used within 3 hours of preparation. Renilla working solution activity is stable for up to 3 hours, but background increases up to 60% after 5 hours at room temperature.

4. Firefly & Renilla Luciferase Single Tube Assay

The protocol below is for manual assay using a single-tube luminometer. If your luminometer is equipped with automatic injectors, they may be used to dispense one or both working solutions into each luminometer tube or well of a multiwell plate according to the instructions for your instrument.

4.1 Set up luminometer with parameters recommended for your instrument for dual luciferase assay. We routinely use integration time of 1 second.

4.2 Add 20 uL of cell lysate into a reaction tube that is compatible with your luminometer.

4.3 Add 100 uL of firefly working solution to the reaction tube and mix by pipetting up and down several times.

Note: Do not vortex the tube, which could cause the firefly reaction mix to coat the upper part of the tube and not effectively mix with the Renilla working solution in step 5.

4.4 Immediately place tube in luminometer and record the firefly luminescence measurement.

4.5 Add 100 uL of Renilla working solution to the same reaction tube and mix by pipetting or vortexing.

4.6 Immediately place tube in luminometer and record the Renilla luminescence measurement.

4.7 Discard the reaction tube, and proceed to the next reaction.

Note: Renilla working solution can be used to measure Renilla luciferase activity in the absence of firefly luciferase, but for direct comparison to samples with both Firefly and Renilla luciferases, you should first add firefly working solution before adding Renilla working solution so the final assay volume remains constant between samples. For determination of Renilla activity only, firefly working solution can be omitted.

NOTE:

Always wear lab coats, gloves and goggles when working with our products although they are low-risk chemicals for R&D only.

注意事项:

1)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。

2) 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。

3 本公司所有产品仅限于专业人员用于生命科学研究，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅。

4) 本公司所有产品必须由合格专业技术人员操作同时佩戴口罩/手套/实验服并遵守生物实验室安全操作规程！

Description

Name	双萤光素酶报告基因检测试剂盒Dual Luciferase Reporter Gene Assay Kit
CAT#		CAS#
Storage#	4℃ Sealed & desiccated & Minimized light exposure & Minimized Freezing And Thawing	Shelf Life#	24 months
Ex(nm)#		Em(nm)#
MW#		Solvent#

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