ADP-Glo? Kinase Assay with PIP2:3PS

产品编号：V1791 品牌：promega 原厂货号：V1791
产品分类：蛋白质检测与分析 > 酶检测和活性分析 > 激酶检测
应用分类：

包装：

1000 assays

运保温度:

到货周期：

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其它组分：

ADP-Glo™ Reagent【子货号：V912A，包装：1 x 5ml，，运保温度：–30°C 到 –10°C】

Kinase Detection Buffer【子货号：V913A，包装：1 x 10ml，，运保温度：–30°C 到 –10°C】

Kinase Detection Substrate【子货号：V914A，包装：1 x 1 each，，运保温度：–30°C 到 –10°C】

Ultra Pure ATP, 10mM【子货号：V915A，包装：1 x 500μl，，运保温度： –65°C】

ADP, 10mM【子货号：V916A，包装：1 x 500μl，，运保温度：–30°C 到 –10°C】

PIP2:3PS Substrate, 1mg/ml PIP2【子货号：V170A，包装：1 x 0.25ml，，运保温度： –65°C】

Lipid Dilution Buffer, 10X【子货号：V180A，包装：1 x 1ml，，运保温度：–30°C 到 –10°C】

MgCl2, 1M【子货号：V330A，包装：1 x 1ml，，运保温度：–30°C 到 –10°C】

描述：

说明		磷脂酰肌醇（ PI ）及其磷酸化衍生物，统称为磷酸肌醇（ Phosphoinositides ），是重要的第二信使，而第二信使作为信号分子在细胞膜重构中起关键作用。它们由一类被称为磷酸肌醇脂质激酶（ Phosphoinositide Lipid Kinases , PIKs ）的激酶家族催化产生。在哺乳动物中，已确定 19 个 PIK 亚型。根据其对磷酸化肌醇环第 3 、 4 、 5 位羟基基团磷酸化偏好，大致分为三个主要的家族：磷酸肌醇 3 - 激酶（ PI3Ks ），磷脂酰肌醇 4- 激酶（ PI4Ks ）和磷酸肌醇磷酸 - 激酶（ PIP5Ks 和 PIP4Ks ）。 Promega 脂质激酶活性检测系统，利用 ADP-Glo Kinase Assay 激酶活性发光检测平台，提供脂质激酶、底物和 ADP-Glo 检测试剂，为脂质激酶反应提供了整套完备试剂。这些试剂包括纯化的人类 I 类 PI3Ks 重组蛋白，优化的反应缓冲液和即用型脂质激酶底物。可单独购买酶，也可购买整套完备试剂盒 PI3K-Glo™ Class I Profiling Kit ，组分包括 PI3Ks （α、β、γ、δ，各 5 μ g ）， PIP2 ： 3PS 脂质激酶底物（ 0.25mg ）和 ADP-Glo ™激酶检测试剂盒 (1,000 次 ) 。底物以冷冻小单层脂质体形式提供，为磷脂酰肌醇（ PI ）或磷酸肌醇 -4,5- 二磷酸（ PIP2 ）与载脂磷脂酰丝氨酸（ PS ）的混合物，两者混合比例为 1:3 。 PI 与 PS 1:3 混合优化后的底物可用于 I 类 PI3Ks 。已证实， PI 与 PS 1:3 混合后的底物可被大多数 PI3K 家族成员识别，可做为通用型 PI 脂质激酶底物。脂质激酶活性检测原理如图 1 所示。将脂质激酶底物（ 1 PI ： 3 PS 或 1 PIP2 ： 3 PS ）、重组脂质激酶和 ATP 共同孵育进行脂质激酶反应，之后用 ADP-Glo ™激酶检测试剂盒检测激酶活性。 ADP-Glo ™激酶检测试剂盒操作分两个步骤： 1 ）激酶反应后，加入 ATP 耗竭试剂终止激酶反应，同时耗尽所有剩余 ATP ，只留下 ADP ； 2 ）加入激酶检测试剂，将 ADP 转变为 ATP ，同时在偶联的萤光素酶 / 萤光素反应中将 ATP 转变为光信号。
特点		• 提供 I 类 PI3Ks 活性检测完整解决方案： • 纯化的人源重组酶，高度特异活性 • 即用型脂质底物（ PI 或 PIP2 ） • 通用反应缓冲液 • 高灵敏度检测 • 选择性卓越：即使 % 底物转化率很低，仍可获得高信噪比 • 结果可靠：动态范围宽、背景低、灵敏度高，减少了数据的不可靠性 • 节约时间：均质检测，简便的 " 加样 - 读数 " 模式 • 避免假阳性结果：特殊配方和发光检测降低了假阳性率 • 节约经费：可轻松升级至 1,536 孔板检测模式，降低每孔检测成本
操作手册		ADP-Glo™ Lipid Kinase Assay Technical Manual
参考文献		• Cantley, L.C. (2002) The phosphoinositide 3-kinase pathway. Science 296 , 1655–7. • Markman, B. et al. (2010) Status of PI3K inhibition and biomarker development in cancer therapeutics. Annals Oncology 21 , 683–91. • Brown, J.R. and Auger, K.R. (2011) Phylogenomics of phosphoinositide lipid kinases: perspectives on the evolution of second messenger signaling and drug discovery. BMC Evolutionary Biology 11 , 1–34. • Hawkins, P.T. et al. (2006) Signalling through Class I PI3Ks in mammalian cells.Biochem. Soc. Trans. 14 , 647–62.

原厂资料：

Phosphatidylinositol (PI) and its phosphorylated derivates, collectively called phosphoinositides, are important second messengers that are critical as signaling molecules and for cellular membrane remodeling. These derivatives are generated by a family of kinases called phosphoinositide lipid kinases (PIKs). Nineteen PIK isoforms have been identified in mammals. Based on their ability to preferentially phosphorylate the hydroxyl group of the inositol ring on position 3, 4 or 5, they have been broadly classified into three major families: phosphoinositide 3-kinases (PI3Ks), phosphoinositide 4-kinases (PI4Ks) and phosphoinositide phosphate-kinases (PIP5Ks and PIP4Ks).

Promega lipid kinase enzymes, substrates and detection systems provide a complete set of reagents for performing phosphoinositide lipid kinase (PIK) reactions using a luminescent ADP-detection platform, the ADP-Glo™ Kinase Assay. The reagents include purified human recombinant proteins of Class I PI3Ks, optimized reaction buffer and ready-to-use lipid kinase substrates. The enzymes are available separately or can be purchased as part of the PI3K-Glo™ Class I Profiling Kit, which contains PI3Ks (α, β, γ and δ; 5μg each), PIP2:3PS Lipid Kinase Substrate (0.25mg) and the ADP-Glo™ Kinase Assay, 1,000 assays. The lipid substrates are supplied as frozen small unilamellar vesicles containing a mixture of phosphatidylinositol (PI) or phosphoinositol-4,5-bisphosphate (PIP2) at a 1:3 ratio with phosphatidylserine (PS) as carrier lipid. A substrate composed of PIP2 and PS at a 1:3 ratio was optimized to use with class I PI3Ks. A substrate composed of PI and PS at a 1:3 ratio was demonstrated to be recognized by the majority of family members and provides a universal PI lipid kinase substrate.

The principle of the lipid kinase assay is illustrated in Figure 1. The lipid kinase reaction is performed by incubating lipid substrate (PI:3PS or PIP2:3PS) with a recombinant enzyme and ATP, and the kinase activity is measured using the ADP-Glo™ Kinase Assay. The ADP-Glo™ Kinase Assay is performed in two steps. After the kinase reaction, an ATP-depletion reagent is added to terminate the lipid kinase reaction and deplete any remaining ATP, leaving only ADP. Next, a detection reagent is added to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be converted to light using a coupled luciferase/luciferin reaction.

Features - Benefits

Employ Complete Solutions for Class I PI3Ks:
Purified human recombinant enzymes with high specific activity.
Ready-to-use lipid substrate (PI or PIP2).
Universal reaction buffer formulation.
Highly sensitive detection assay.
Observe Excellent Selectivity: High signal-to-background ratios even at low % conversion of substrate.
Obtain Reliable Results: The broad dynamic range, low background and excellent sensitivity result in less ambiguous data.
Save Time: Homogeneous assay with simple "add-and-read" format.
Avoid False Hits: The special formulation and luminescent signal results in low false-hit rate.
Save Money: Easily scalable to 1,536-well format, reducing cost per well.

References

Cantley, L.C. (2002) The phosphoinositide 3-kinase pathway. Science 296, 1655–7.
Markman, B. et al. (2010) Status of PI3K inhibition and biomarker development in cancer therapeutics. Annals Oncology 21, 683–91.
Brown, J.R. and Auger, K.R. (2011) Phylogenomics of phosphoinositide lipid kinases: perspectives on the evolution of second messenger signaling and drug discovery. BMC Evolutionary Biology 11, 1–34.
Hawkins, P.T. et al. (2006) Signalling through Class I PI3Ks in mammalian cells. Biochem. Soc. Trans. 14, 647–62.

注意事项：

PI3K 重组酶（ Recombinant PI3K Enzymes ）：将 PI3K 重组酶储存于 –65°C 以下。首次使用，快速融化置于冰上，根据每次实验用量将酶分装成小份，并立即冻存分装管。避免反复冻融。

脂质底物：将脂质底物储存于 –65°C 。使用前，室温融化至底物完全平衡到室温。涡旋震荡，充分混合至少 1 分钟。融化后的脂质底物可在室温保存至少 6 小时（ 15-30 ℃），或储存在 2-10 ℃一个星期。

缓冲液：将 5X PI3K Reaction Buffer ， 10X Lipid Dilution Buffer 和 1M MgCl2 储存于 –30°C 到 –10°C 。

ADP-Glo™ 检测试剂盒：收到试剂盒后，将 ATP 取出并储存于 –65°C 以下，其余组分储存于 –30°C 到 –10°C 。使用前，将所有组分在室温下彻底融化。融化后，将每种组分充分混匀。由于 ATP 反复冻融容易水解，请将其分装成小份储存于 –65°C 以下。激酶检测试剂（激酶检测缓冲液 + 底物）和 ADP- Glo™ 试剂也应分装成小份储存于 –30°C 到 –10°C 。为方便使用，这两种试剂在室温下存放 24 小时内不会产生信号衰减。