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PhosphoWorks 荧光ATP检测试剂盒

英文名称:PhosphoWorks™ Fluorimetric ATP Assay Kit
产品参数
Ex (nm)-Em (nm)-
分子量-溶剂-
存储条件-
产品概述

PhosphoWorks 荧光ATP检测试剂盒是美国AAT Bioquest生产的用于检测ATP的试剂盒,三磷酸腺苷(ATP)在细胞能量,代谢调节和细胞信号传导中起着基本作用。它被称为细胞内能量转移的“货币分子单位”,以驱动活细胞中的许多过程和化学合成。 ATP也是细胞通讯的信号分子,在DNA和RNA合成中起重要作用。 AAT Bioquest提供了多种生物发光测定试剂盒,可通过重组萤火虫荧光素酶(目录号21610和21609)确定ATP的纳摩尔(nM)范围。这些试剂盒需要酶标仪,通常用于细胞活力或细胞毒性测定。 PhosphoWorks 荧光ATP分析试剂盒基于一系列ATP诱导的酶偶联反应,产生过氧化氢,该过氧化氢用我们的Amplite Red底物通过分光光度法定量。该测定法可在100 µL反应体积中检测到约0.4 µM ATP,而不受ADP和AMP的干扰。它为测定生物样品中的ATP水平提供了一种强大,简单且方便的测定方法。 PhosphoWorks 荧光ATP分析是我们基于荧光素酶的ATP分析试剂盒的补充。百萤生物是AAT Bioquest的中国代理商,为您提供优质的PhosphoWorks 荧光ATP检测试剂盒。

 

适用仪器


荧光酶标仪  
Ex: 540 nm
Em: 590 nm
Cutoff: 570 nm
推荐孔板: 纯黑色孔板
实验方案

样品实验方案

简要概述

1.准备ATP工作溶液(50 µL)
2.添加ATP标准液或测试样品(50 µL)
3.在室温下孵育10-30分钟
4.监测Ex / Em = 540/590 nm(截止= 570 nm)的荧光增加

 

溶液配制

1.制备储备溶液

所有未使用的储备溶液应分为一次性使用的等分试样,并在制备后储存在-20°C下。 避免重复冻融循环。
1.1Amplite 红色底物储备液(200X):
将30 µL DMSO(组分E)加入小瓶Amplite Red Substrate(组分A)中,制成200X Amplite Red Substrate储备液。

1.2ATP标准溶液(10mM):
将0.5 mL的ddH2O加入到ATP标准溶液(组分D)的小瓶中,制成10 mM ATP标准溶液。

 

2.制备标准溶液

ATP标准

将10 µL的10 mM ATP标准溶液添加到990 µL 1X PBS缓冲液中,以生成100 µM ATP标准溶液(AS7)。 取100 µM ATP标准溶液(AS7)并进行1:3连续稀释,以使用1X PBS缓冲液获得系列稀释的ATP标准溶液(AS6-AS1)。

 

3.制备工作溶液

3.1将5毫升测定缓冲液(组分C)添加到酶混合瓶(组分B)中,并充分混合。

3.2在Enzyme Mix瓶中加入25 µL 200X AmpliteTM Red Substrate储备液,充分混合以制成APT工作溶液。

 

样品示例及操作

表1.黑色96孔微孔板中ATP标准品和测试样品的布局。 AS = ATP标准品(AS1-AS7,0.14至100 µM),BL =空白对照,TS =测试样品。

BL BL TS TS
AS1 AS1 ... ...
AS2 AS2 ... ...
AS3 AS3    
AS4 AS4    
AS5 AS5    
AS6 AS6    
AS7 AS7    

表2.每个孔的试剂组成

容积 试剂
AS1-AS7 50ul 连续稀释(0.14至100 µM)
BL 50ul 1 X PBS缓冲液
TS 50ul 测试样品

1.根据表1和2中提供的布局,准备ATP标准品(AS),空白对照(BL)和测试样品(TS)。对于384孔板,每孔使用25 µL试剂,而不是50 µL。

2.将50 µL ATP工作溶液添加到ATP标准液,空白对照和测试样品的每个孔中,以使ATP总测定体积为100 µL /孔。 对于384孔板,将25 µL ATP工作溶液添加到每个孔中,总体积为50 µL /孔。

3.避光保存,室温下孵育反应10-30分钟。

4.使用荧光酶标仪在Ex / Em = 540/590 nm(截止= 570 nm)处监视荧光增加。

 

参考文献

ATP-based cell viability assay is superior to trypan blue exclusion and XTT assay in measuring cytotoxicity of anticancer drugs Taxol and Imatinib, and proteasome inhibitor MG-132 on human hepatoma cell line HepG2
Authors: E. Nowak
Journal: Clin Hemorheol Microcirc (2018): 327-336

High-content image analysis (HCIA) assay has the highest correlation with direct counting cell suspension compared to the ATP, WST-8 and Alamar blue assays for measurement of cytotoxicity
Authors: H. Tahara
Journal: J Pharmacol Toxicol Methods (2017): 92-99

High throughput cell-based assay for identification of glycolate oxidase inhibitors as a potential treatment for Primary Hyperoxaluria Type 1
Authors: Mengqiao Wang, Miao Xu, Yan Long, Sonia Fargue, Noel Southall, Xin Hu, John C McKew, Christopher J Danpure, Wei Zheng
Journal: Scientific Reports (2016)

NT1014, a novel biguanide, inhibits ovarian cancer growth in vitro and in vivo
Authors: Lu Zhang, Jianjun Han, Amanda L Jackson, Leslie N Clark, Joshua Kilgore, Hui Guo, Nick Livingston, Kenneth Batchelor, Yajie Yin, Timothy P Gilliam
Journal: Journal of Hematology & Oncology (2016): 91

The Different Effects of Atorvastatin and Pravastatin on Cell Death and PARP Activity in Pancreatic NIT-1 Cells
Authors: Ya-Hui Chen, Yi-Chun Chen, Chin-San Liu, Ming-Chia Hsieh
Journal: Journal of Diabetes Research (2016)

BPA-induced DNA hypermethylation of the master mitochondrial gene PGC-1α contributes to cardiomyopathy in male rats
Authors: Ying Jiang, Wei Xia, Jie Yang, Yingshuang Zhu, Huailong Chang, Juan Liu, Wenqian Huo, Bing Xu, Xi Chen, Yuanyuan Li
Journal: Toxicology (2015): 21--31

Combination of immunoprecipitation (IP)-ATP_Glo kinase assay and melanogenesis for the assessment of potent and safe PAK1-blockers in cell culture
Authors: B. C. Nguyen
Journal: Drug Discov Ther (2015): 289-95

Glutamine promotes ovarian cancer cell proliferation through the mTOR/S6 pathway
Authors: Lingqin Yuan, Xiugui Sheng, Adam K Willson, Dario R Roque, Jessica E Stine, Hui Guo, Hannah M Jones, Chunxiao Zhou, Victoria L Bae-Jump
Journal: Endocrine-related cancer (2015): 577--591

JQ1 suppresses tumor growth through downregulating LDHA in ovarian cancer
Authors: Haifeng Qiu, Amanda L Jackson, Joshua E Kilgore, Yan Zhong, Leo Li-Ying Chan, Paola A Gehrig, Chunxiao Zhou, Victoria L Bae-Jump
Journal: Oncotarget (2015): 6915

Loss of histone deacetylase Hdac1 disrupts metabolic processes in intestinal epithelial cells
Authors: Alexis Gonneaud, Naomie Turgeon, Frančois-Michel Boisvert, Frančois Boudreau, Claude Asselin
Journal: FEBS letters (2015): 2776--2783

 

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