- 中文名称
Kinase Assay Kit, BioAssay
- 英文名字
- Kinase Assay Kit, BioAssay
- 供应商
- USBiological
- 产品货号
- K1774-10
- 产品报价
- ￥1/96Tests
- 产品说明书
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- 产品新闻
- 背景资料
- USBiological品牌是全球有名的抗原抗体和生化试剂供应商，生产世界上种类最多的抗体，用于Western Blot、免疫沉淀、免疫荧光、免疫组化和流式细胞术等多种检测方法。武汉艾美捷科技作为USBiological品牌中国区域总代理，是行业中少有的致力于服务客户，帮助客户，且拥有独立的专业销售团队、技术支持团队、市场营销团队、进出口报关团队的高科技生物企业。可以为您提供及时的咨询响应，专业的产品和解决方案支持，稳健快捷的交货周期，优质放心的售后服务。我们致力于为您提供有价值的产品和服务，在意您的成功！
- 产品描述
- Kinases, also known as phosphotransferases, constitute a large family of enzymes that transfer phosphate groups from the high-energy donor molecule ATP, to their specific substrates. Kinases are known to regulate the majority of cellular processes. The largest group of this family is the protein kinases. So far, 518 different kinases have been identified in humans and up to 30% of human proteins are modified by these kinases. The enormous diversity and their key role in cellular signaling make them ideal targets for drug developments.Kinase Assay Kit provides a simple and rapid method for assaying kinase activity and high-throughput screening for kinase inhibitors. This homogeneous microplate-based assay involves incubating the kinase with a single working reagent, in which ADP is enzymatically converted to ATP and pyruvate, which is quantified using a fluorometric (530nm/590nm) assay method.Key Features:Safe. Non-radioactive assay.Sensitive and accurate. As low as 0.01 U/L kinase can be quantified.Homogeneous and convenient. Mix-incubate-measure type assay. The whole assay involves adding a single working reagent and incubation for 10 min at room temperature.Robust and amenable to HTS: Assay can tolerate up to 300uM ATP and 10% dimethylsulfoxide (DMSO). Z’ factors of > 0.6 are routinely observed in 96/384-well plates. Can be readily automated on HTS liquid handling systems for tens of thousands of assays per day.Applications:Kinase activity assays and HTS for kinase inhibitors.ADP determination in cells and other biological samples.Kit Contents:Reagent A: 10ml Reagent B: 10mlAssay Buffer: 25ml Standard: 100ul 3mM ADPStorage conditions: store all reagents at -20°C. This product is shipped on dry ice. Shelf life: 6 months after receipt.Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.Assay Procedure:This kit is sufficient for 400 assays in 384-well plate (200 assays in 96-well plate). Use black flat-bottom plates. Prior to assay, bring all reagents to room temperature. Assays in duplicate wells are recommended. Interference: thiols (b-mercaptoethanol, dithioerythritol etc) at > 10uM interfere with this assay and should be avoided. Kinase Activity Assay in 384-well Plate1. Kinase Reaction. Users should provide their own enzyme, ultra-pure ATP and substrate. Set up 20ul reaction mixture containing the kinase, ATP and substrate in the provided Assay Buffer (pH 7.0) or any suitable kinase buffer. Set up a Blank Control that contains ATP and substrate but no enzyme. Incubate at desired temperature for desired period of time e.g. 30 min.2. Standards. Prepare 900ul 10uM ADP Premix by mixing 3ul 3mM Standard and 897ul distilled water. Dilute standard as follows. Transfer 20ul standards into separate wells of the plate.No Premix+H2O Vol (ul) ADP (uM)1 100ul+0ul 100 102 60ul+40ul 100 63 30ul+70ul 100 34 0ul+100ul 100 03. ADP Detection. Prepare enough Working Reagent for each well by mixing 25ul Reagent A and 25ul Reagent B. Add 40ul Working Reagent to each assay well. Tap plate to mix. Incubate at room temperature for 10 min.4. Read fluorescence intensity at lexc=530nm and lem=590nm. Calculate kinase activity,Kinase Activity =DFSAMPLESlope × tx (U/L)20ulVol (ul)where DFSAMPLE=(fluorescence intensity of sample well–fluorescence of the blank well), slope is the slope of the ADP standard curve. t is the kinase reaction time (e.g. 30 min). Vol is the volume (ul) of kinase added to the 20ul reaction.Note: typical kinase assays use 100 to 200uM ATP. If DFSAMPLE > (F10uM ADP–FH2O), dilute enzyme in assay buffer. Repeat the assay, multiply the results by the dilution factor n. This will ensure that the initial rate is measured at < 10% ATP conversion.High-throughput Screen for Kinase InhibitorsFor inhibitor screens, test compounds are usually pre-incubated with kinase for 10 to 30 min, prior to adding ATP/substrate to initiate kinase reaction. After a 30-min kinase reaction, the produced ADP is quantified using the Fluorometric Assay:. The fluorescence intensity will be decreased in the presence of an inhibitor.1. Controls and compounds. Use a known kinase inhibitor (e.g. staurosporine) as a positive control. Alternatively, “no enzyme” wells can serve as a positive control. Use the same volume of the compound solvent (e.g. DMSO) as an inhibitor negative control. Example: transfer 5ul test compound, control inhibitor and negative control (e.g. DMSO) to appropriate wells, add 10ul enzyme solution to all assay wells. Apply an “in-well” mixing step. Incubate for 10 to 30 min.2. Kinase Reaction. Add 5ul mixture containing ATP and the kinase substrate to each assay well. Mix well. Incubate for desired period of time (e.g. 30 min).3. ADP Detection. Prepare enough Working Reagent by mixing 25ul Reagent A and 25ul Reagent B. Add 40ul Working Reagent to each well and mix well. Incubate for 10 min.4. Read fluorescence intensity at lexc=530nm and lem=590nm.
- 产品特点
- 针对Kinase Assay Kit, BioAssay该产品的特点优势，欢迎查阅官网提供的产品说明书。
- 保存建议
- 建议收到Kinase Assay Kit, BioAssay产品后将其置于-20°C保存。
- 其他
- Usbiological公司是美国著名的抗体和生化试剂供应商，生产世界上种类最多的抗体，用于Western Blot、免疫沉淀、免疫荧光、免疫组化和流式细胞术等多种检测方法。Usbiological公司现已拥有超过50,19234种抗体、抗原和生化产品，为科研用户提供了诸多超值选择。
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- 注意
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该页面的中文产品信息的翻译，仅供参考。准确的产品信息请以厂家的英文说明书为准。下单前，请浏览说明书确认。
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