Renilla luciferase assay kit

真核基因表达调控研究常用的方法是进行报告基因的检测，生物发光法又是报告基因检测最常用的有效手段。萤光素酶能催化底物萤光素的转化，并发射出光子。该产品为海肾萤光素酶报告基因在哺乳动物细胞中的表达提供快速、灵敏、稳定的检测方法。 产品特点： 1. 快速：细胞裂解在 10-15 min 内完成； 2. 方便：试剂易于配制，样品检测步骤简单。 使用方法： 1. 细胞裂解 （1）将培养基移除，加 PBS 轻轻洗涤（贴壁细胞可直接进行此操作，悬浮细胞要离心收集细胞）。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（裂解产物不能多次反复冻融）。 （2）将充分裂解后的裂解产物，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。 2. 工作液配制 （1）将所有组分恢复至室温。 （2）用组分 B 将组分 C 稀释成海肾萤光素酶工作液，稀释方法为将 1 µL C 组分加入到 49 µL B 组分中。 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）将细胞裂解产物按照 20~100 µL 的体积加入测量管中（保持每次样品量一致）。1×Lysis Buffer 为空白对照。 （3）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）。 注：海肾萤光素酶工作液不能长时间保存，现用现配，一次性使用完毕。 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. 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. 海肾萤光素酶催化的生物发光的最强波长为 480 nm，为防止孔间干扰，建议使用白色不透光孔板； 4 ..B组分建议根据实验需求进行小批量分装； 5.建议现配现用，避免反复冻融； 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 The commonly used method of eukarYOtic gene expression regulation research is the detection of reporter genes, and bioluminescence is the most commonly used and effective means of reporter gene detection. Luciferase can catalyze the conversion of the substrate luciferin and emit photons. This product provides a rapid, sensitive and stable detection method for the expression of Renilla luciferase reporter gene in mammalian cells. Product characteristic： 1.Rapid : Cell lysis was completed within 10-15 min ； 2.Convenience : The reagent is easy to prepare, and the sample detection steps are simple； Instruction： 1. Cell lysis ( 1 ) Remove the culture medium and gently wash with PBS ( adherent cells can be directly performed this operation, suspension cells should 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 after full pyrolysis were centrifuged at 10000-15000 rpm for 3-5 min. After centrifugation, the supernatant was moved into a new EP tube for subsequent detection. 2. Preparation of working fluid ( 1 ) Restore all components to room temperature. ( 2 ) Dilute component C into renilla luciferase working solution with component B, and the dilution method is to add 1 μL C component to 49 μL B component. 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 ) The cell lysis products were added to the measuring tube according to the volume of 20 ~ 100 μL ( keep the same amount of samples each time ). 1 × Lysis Buffer was blank control. ( 3 ) 100 μL renilla luciferase working solution was added to determine the RLU ( Relative light unit ) value ( Shaking mixing function is recommended for microplate reader ). Note : The renilla luciferase working solution cannot be stored for a long time. It is now ready for use and is used once. Component： RenillaLuciferase Lysis Buffer；RenillaLuciferase Assay Buffer；Coelenterazine Matters needing attention： Scope of application： Matters needing attention： 1.Please instantaneously centrifuge the product to the bottom of the tube before use, and then carry out subsequent experiments ; 2.Due to the influence of temperature on the enzyme reaction, the sample and reagent should be measured after reaching room temperature. 3.The strongest wavelength of bioluminescence catalyzed by renilla luciferase is 480 nm, in order to prevent interference between holes, it is recommended to use white opaque orifice plate ; 4.. B component is recommended to carry out small batch packing according to the experimental requirements ; 5.It is recommended to use it now to avoid repeated freezing and thawing ; 6.For your safety and health, please wear experimental clothes and wear disposable gloves. Scope of application： Study on gene expression regulation and promoter

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