NAD/NADH-Glo? Assay

产品编号：G9071 品牌：promega 原厂货号：G9071
产品分类：生化和细胞分析 > 细胞分析试剂盒 > 细胞代谢检测试剂盒
应用分类：

包装：

10ml

运保温度:

-20℃

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其它组分：

Reductase【子货号：G884A，包装：1 x 55μl，，运保温度：-20℃】

Reductase Substrate【子货号：G885A，包装：1 x 55μl，，运保温度：-20℃】

NAD Cycling Enzyme (lyophilized)【子货号：G887A，包装：1 x 1 vial，，运保温度：-20℃】

NAD Cycling Substrate【子货号：G888A，包装：1 x 1.25ml，，运保温度：-20℃】

Luciferin Detection Reagent【子货号：V859A，包装：1 x 1 each，，运保温度：-20℃】

Reconstitution Buffer【子货号：V865A，包装：1 x 10ml，，运保温度：-20℃】

描述：

说明

NAD/NADH-Glo ™ Assay 是一种均质的化学发光检测试剂盒，能够检测总的氧化型和还原型烟酰胺腺嘌呤二核苷酸（ NAD + 和 NADH ），并确定 NAD + 和 NADH 在生物样品或特定酶学反应中的比值。 NAD 循环酶用于将 NAD + 转换为 NADH 。在 NADH 存在时，试剂盒所提供的还原酶将 proluciferin （还原酶底物）还原成萤光素。萤光素通过 Ultra-Glo ™重组萤光素酶定量，反应产生的光信号正比于样品中的 NAD + 和 NADH 的量。 NAD 循环酶和还原酶所驱动的 NAD + 和 NADH 循环增加了检测的灵敏度和选择性，使得试剂盒只检测非磷酸化的 NAD + 和 NADH ，而不会检测磷酸化形式的 NADP + 和 NADPH 。

NAD 循环酶、还原酶和萤光素酶反应是通过将等体积的 NAD/NADH-Glo ™ Detection Reagent 加入 NAD + - 或 NADH- 样品中而被启动，检测试剂中包含 NAD 循环酶和底物、还原酶、还原酶底物和 Ultra-Glo ™ rLuciferase ，同时检测试剂中存在的去污剂可将细胞裂解，这样通过加入单一试剂即可检测多孔板中细胞内总 NAD + 和 NADH 的含量。另外，我们也提供了可单独测量 NAD + 和 NADH 、计算 NAD + 和 NADH 比例的操作手册。简单的“加样 - 混合 - 检测”操作方案和灵活、可扩展的检测方法使得 NAD/NADH-Glo ™ Assay 非常适合高通量筛选小分子化合物对 NAD 和 NADH 水平的影响。

特点

• 高灵敏度 ：可直接检测细胞孔中总 NAD+ 和 NADH 含量，对细胞数量要求更少，无需样品制备。

• 一步法、均质检测 ：只需在 96 或 384 孔细胞培养板中加入单一检测试剂，即可直接检测总 NAD + 和 NADH 。另外还提供简单的细胞培养板内分别测定 NAD+ 和 NADH 的操作方案。

• 线性范围宽 ：可检测 10nm 至 400nm 的 NAD + 或 NADH 。该试剂盒检测 100nM 的信号值高于背景值五倍以上，且 window （最大信噪比）≥ 100 。

• 易于自动化 ：操作方案兼容自动化和高通量检测。反应体系可调，支持 96 、 384 和 1536 孔板检测。

• 可靠、具有良好的可重复性 ： NAD/NADH-Glo ™ Assay 在通常实验中的 Z` 因子值 >0.7 。

原厂资料：

The NAD/NADH-Glo™ Assay is a bioluminescent, homogeneous single-reagent-addition assay for detecting total oxidized and reduced nicotinamide adenine dinucleotides (NAD+ and NADH, respectively) and determining their ratio in biological samples or in defined enzyme reactions. An NAD Cycling Enzyme is used to convert NAD+ to NADH. In the presence of NADH, the provided reductase enzyme reduces a proluciferin reductase substrate to form luciferin. Luciferin then is quantified using Ultra-Glo™ Recombinant Luciferase, and the light signal produced is proportional to the amount of NAD+ and NADH in the sample. Cycling between NAD+ and NADH by the NAD Cycling Enzyme and Reductase increases assay sensitivity and provides selectivity for the nonphosphorylated NAD+ and NADH compared to the phosphorylated forms NADP+ and NADPH.

The NAD Cycling Enzyme, Reductase and luciferase reactions are initiated by adding an equal volume of NAD/NADH-Glo™ Detection Reagent, which contains NAD Cycling Enzyme and Substrate, Reductase, Reductase Substrate and Ultra-Glo™ Recombinant Luciferase, to an NAD+- or NADH-containing sample. Detergent present in the reagent lyses cells, allowing detection of total cellular NAD+ and NADH in a multiwell format with addition of a single reagent. An accessory protocol is provided to allow separate measurements of NAD+ and NADH, and calculation of the NAD+ to NADH ratio. The simple add-mix-read protocol and scalable assay chemistry make the NAD/NADH-Glo™ Assay well suited to monitor effects of small molecule compounds on NAD and NADH levels in high-throughput formats.

Features - Benefits

High Sensitivity: High sensitivity of the assay enables detection of total NAD+ and NADH directly in the wells. Fewer cells are required, with no sample preparation.
Homogeneous, One-Step Protocol: Total NAD+ and NADH is measured directly in wells of a 96- or 384-well cell culture plate with one reagent addition. A simple in-plate protocol is provided for individual NAD+ and NADH measurements.
Large Assay Window: The NAD/NADH-Glo™ Assay detects 10nM to 400nM NAD+ or NADH. The assay detects 100nM with a signal higher than fivefold over background and an assay window (maximum signal-to-background ratio) of ≥100.
Automation Compatible: The assay is compatible with automated and high-throughput protocols. Reactions are scalable and can be performed at low volumes in 96-, 384- and 1536-well plates.
Reliability and Reproducibility: The NAD/NADH-Glo™ Assay routinely yields Z´ factors >0.7.
Luminescence-Based NAD+ and NADH Detection: The luminescent format avoids fluorescent interference due to reagents and test compounds sometimes seen in fluorescent assays.

Applications

Monitoring changes in cellular levels of total NAD+ and NADH.
Determining NAD/NADH ratios.
Monitoring the effects of small molecule compounds on NAD+ and NADH levels in enzymatic reactions or directly in cells in high-throughput formats.

注意事项：

G9071 contains sufficient reagents to perform 100 reactions in 96-well plates (100μl of sample + 100μl of NAD/NADH-Glo™ Detection Reagent), 400 assays in 384-well plates (25μl of sample + 25μl of NAD/NADH-Glo™ Detection Reagent) or 2,000 assays in low-volume 384-well plates (5μl of sample + 5μl of NAD/NADH-Glo™ Detection Reagent). Assay volumes can be varied depending on plate format as long as a 1:1 ratio of sample to NAD/NADH-Glo™ Detection Reagent is maintained.