AMP-Glo? Assay

产品编号：V5011 品牌：promega 原厂货号：V5011
产品分类：生化和细胞分析 > 生化分析试剂盒 > 其它
应用分类：

包装：

1000 Assays

运保温度:

-30 至 -10 ℃

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其它组分：

AMP-Glo™ Reagent I【子货号：V503A，包装：1 x 5ml，，运保温度：-30 至 -10 ℃】

AMP-Glo™ Reagent II【子货号：V504A，包装：1 x 100μl，，运保温度：-30 至 -10 ℃】

Kinase-Glo® One Solution【子货号：V505A，包装：1 x 10ml，，运保温度：-30 至 -10 ℃】

AMP, 10mM【子货号：V506A，包装：1 x 100μl，，运保温度：-30 至 -10 ℃】

Ultra Pure ATP, 10mM【子货号：V915A，包装：1 x 500μl，，运保温度：-30 至 -10 ℃】

描述：

说明		AMP-Glo™ Assay （ AMP-Glo ™检测试剂盒）是一种均质的、利用生物发光原理检测 AMP 的方法，可用于检测任何产生 AMP 的生化反应。该多用途系统，可以检测多种酶，如 cAMP- 特异性磷酸二酯酶，氨基酰 -tRNA 合成酶， DNA 连接酶和泛素连接酶或由 AMP 调控的酶。 AMP-Glo ™检测试剂盒可在多种规格的反应板中定量监测生化反应中的 AMP 浓度，并可用于高通量检测。该试剂盒稳定的发光信号，因而不需使用带有进样器的生物发光仪，并允许多个微孔板的批量处理。 ATP 存在或不存在的情况下，该方法均可定量检测反应产生的 AMP 。该试剂盒包含两种试剂：试剂 1. 终止酶促反应的 AMP 产生并同时除去 ATP ，将 AMP 转换成 ADP ；试剂 2. 将 ADP 转换成 ATP ， ATP 参与萤光素 / 萤光素酶反应产生发光信号。该试剂盒还适用于检测不使用 ATP 作为底物的生化反应，如 cAMP- 依赖性磷酸二酯酶（ PDE ）和细菌的 DNA 连接酶。 AMP-Glo ™检测试剂盒具有很宽的动态范围，在低底物转化情况下仍产生很强的信号，使得它非常适合于筛选低活性酶。该试剂盒产生的假阳性率最低，Z` 值大于 0.7 。
特点		• 底物转换率很低的情况下仍可获得高信号强度：测量的酶活性，更接近生理条件，非常适合于低活性的酶。 • 灵敏，对极低浓度的 AMP 也具敏感性：节省成本，相比其他试剂盒，消耗更少的酶。 • 通用：可检测几乎任何 AMP 生成酶，仅用单一平台即可以筛选更广泛的酶。 • 在很宽范围的起始底物浓度下，精确测量 AMP 水平：真实反映酶活性，非常适合测量抑制剂对酶活性的影响。 • 发光法读数：相比基于荧光的方法，较不易受化合物库干扰。
应用		• 筛选化合物对靶酶的影响。 • 可用于多种酶： • 泛素连接酶 • cAMP- 特异性磷酸二酯酶 • 氨酰 -tRNA 合成酶 • DNA 连接酶 • poly(A) 去乙酰化酶 • 去甲基化酶（间接法）
操作手册		AMP-Glo™ Assay Technical Manual
注意		AMP-Glo™ 检测试剂盒提供足够的试剂以进行说明书中指定数量的小量体积 ——384 孔板检测（ 5μl 反应酶： 5μl AMP-Glo™ I Reagent ： 10μl AMP Detection Solution ）。该系统还可用于标准的 96 孔板反应（前述溶液比例为： 25μl ： 25μl ： 50μl ）。
储存条件		储存于 -30 至 -10 ℃。使用前， AMP-Glo ™ Reagent II 应于冰上融化，将其余组分在室温下完全融化。使用前将各组分彻底混匀。应将 Kinase-Glo ® One Solution 分装成小份储存于 -20 ℃。有效期见产品标签。

参考文献

• Zhang, L. et al. (2008) Cyclic nucleotide phosphodiesterase profiling reveals increased expression of phosphodiesterase 7B in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA105 , 19531–6.

• Guo, M. et al. (2010) New functions of aminoacyl-tRNA synthetases beyond translation. Nature Rev. Mol. Cell. Biol. 11 , 668–74.

• Mills, S.D. et al. (2011) Novel bacterial NAD+-dependent DNA ligase inhibitors with broad-spectrum activity and antibacterial efficacy in vivo. Antimicrob. Agents Chemother. 55 , 1088–96.

• Robinson, P.A. and Ardley, H.C. (2011) Ubiquitin-protein ligases. J. Cell Sci. 117 , 5191–4.

• Galperin, M.Y. and Koonin, E.V. (1997) A diverse superfamily of enzymes with ATP-dependent carboxylate-amine/thiol ligase activity. Protein Science 6 , 2639–43.

• Steinberg, G.R. and Kemp, B.E. (2009) AMPK in health and disease. Phyisol. Rev. 89 , 1025–78.

原厂资料：

The AMP-Glo™ Assay is a homogeneous assay that generates a luminescent signal from any biochemical reaction that produces AMP as a reaction product. This versatile system can measure the activity of a broad range of enzymes, such as cyclic AMP-specific phosphodiesterases, aminoacyl-tRNA synthetases, DNA ligases and ubiquitin ligases or enzymes modulated by AMP. The AMP-Glo™ Assay is designed to quantitatively monitor the concentration of AMP in a biochemical reaction in a wide range of plate formats, including high-throughput formats. The stable luminescent signal of the assay eliminates the need for an injector-equipped luminometer and allows batch-mode processing of multiple plates. The assay can be used to determine the AMP produced either in the presence or absence of ATP as a substrate.

The assay contains two reagents: one to terminate the AMP-generating enzymatic reaction and simultaneously remove ATP and convert AMP produced into ADP, and a second reagent that converts the ADP to ATP followed by conversion of the ATP into a luminescent signal using the luciferin/luciferase reaction. The assay also is well suited for monitoring AMP produced in biochemical reactions catalyzed by enzymes that do not use ATP as a substrate, such as cAMP-dependent phosphodiesterases (PDE) and bacterial DNA ligases.

The AMP-Glo™ Assay has a high dynamic range and produces a strong signal at low substrate conversion, making it well suited for screening low activity enzymes. The assay produces minimal false hits and Z´ values greater than 0.7.

Features - Benefits

High Signal Strength at Low Substrate Conversion: Measure enzyme activity that more closely mimics physiological conditions—very well suited for low-activity enzymes.
Sensitive to Low Concentrations of AMP: Requires less enzyme than other assays; cost savings.
Universal: Use the assay with virtually with any AMP-producing enzyme—enables screening of a wider range of enzymes using a single platform.
Accurately Measures AMP Levels at a Wide Range of Starting Substrate Concentrations: Activity measured truly reflects enzyme activity and is well suited for measuring the effects of inhibitor on enzyme activity.
Luminescent Readout: Much less susceptible to interference from library compounds than fluorescent-based methods.

Applications

Screen library compounds for effects on target enzymes.
Use with a wide range of enzymes:
- ubiquitin ligases
- cyclic AMP-specific phosphodiesterases
- aminoacyl-tRNA synthetases
- DNA ligases
- poly(A) deadenylases
- demethylases (indirect method)

Notes

The AMP-Glo™ Assays contain enough reagent to perform the indicated number of reactions in a low-volume 384-well plate format using 5μl of reaction enzyme, 5μl of AMP-Glo™ I Reagent and 10μl of AMP Detection Solution. The system also can be used in standard 96-well plates (25μl:25μl:50μl).

References

Zhang, L. et al. (2008) Cyclic nucleotide phosphodiesterase profiling reveals increased expression of phosphodiesterase 7B in chronic lymphocytic leukemia.Proc. Natl. Acad. Sci. USA 105, 19531–6.
Guo, M. et al. (2010) New functions of aminoacyl-tRNA synthetases beyond translation. Nature Rev. Mol. Cell. Biol. 11, 668–74.
Mills, S.D. et al. (2011) Novel bacterial NAD+-dependent DNA ligase inhibitors with broad-spectrum activity and antibacterial efficacy in vivo. Antimicrob. Agents Chemother. 55, 1088–96.
Robinson, P.A. and Ardley, H.C. (2011) Ubiquitin-protein ligases. J. Cell Sci. 117, 5191–4.
Galperin, M.Y. and Koonin, E.V. (1997) A diverse superfamily of enzymes with ATP-dependent carboxylate-amine/thiol ligase activity. Protein Science 6, 2639–43.
Steinberg, G.R. and Kemp, B.E. (2009) AMPK in health and disease. Phyisol. Rev. 89, 1025–78.

注意事项：

AMP-Glo™ 检测试剂盒提供足够的试剂以进行说明书中指定数量的小量体积 ——384 孔板检测（ 5μl 反应酶： 5μl AMP-Glo™ I Reagent ： 10μl AMP Detection Solution ）。该系统还可用于标准的 96 孔板反应（前述溶液比例为： 25μl ： 25μl ： 50μl ）。

储存于 -30 至 -10 ℃。使用前， AMP-Glo ™ Reagent II 应于冰上融化，将其余组分在室温下完全融化。使用前将各组分彻底混匀。应将 Kinase-Glo ® One Solution 分装成小份储存于 -20 ℃。有效期见产品标签。