HDAC-GloTM I/II Screening System

产品编号：G6430 品牌：promega 原厂货号：G6430
产品分类：生化和细胞分析 > 其它细胞分析试剂盒 > 去乙酰化检测试剂盒
应用分类：

包装：

10ml

运保温度:

HeLa Nuclear Extract 储存于-70℃,其他组分-20℃

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其它组分：

HDAC-Glo™ I/II Buffer【子货号：G648A，包装：1 x 25ml，，运保温度：-20℃】

HDAC-Glo™ I/II Substrate【子货号：G649A，包装：1 x 1 vial，，运保温度：-20℃】

Developer Reagent【子货号：G653A，包装：1 x 10μl，，运保温度：-20℃】

Trichostatin A【子货号：G656A，包装：1 x 10μl，，运保温度：-20℃】

HeLa Nuclear Extract【子货号：G657A，包装：1 x 10μl，，运保温度：-70℃】

描述：

说明

HDAC-Glo^™ I/II Assays and Screening Systems （ HDAC-Glo^™ I/II 检测试剂盒及筛选系统）是单试剂均质发光检测试剂盒，用于检测来源于细胞、细胞提取物或纯化酶类的第 I 类和第 II 类组蛋白去乙酰酶（ HDAC ）的相对活性。 HDAC-Glo^™ I/II 检测试剂盒使用一种可渗透进入活细胞的乙酰化发光肽底物，该底物能被 HDAC 的活性去乙酰化。氨基萤光素肽底物的去乙酰化可通过一个偶联的酶反应来检测；在这个反应中，显影试剂（Developer Reagent ）中的蛋白酶可将去乙酰化的肽从氨基萤光素底物上切除下来，释放的氨基萤光素可用 Ultra-Glo^™ 重组萤火虫萤光素酶反应来定量。该检测法可在 15–45 分钟内完成，无需样品操作。 HDAC 介导的发光信号持久，半衰期大于 3 个小时，可以用多孔板进行批量操作。 HDAC 检测试剂盒可广泛应用于第 I 类和第 II 类酶。

曲谷菌素 A （Trichostatin A ）包含在 HDAC-Glo^™ I/II 筛选系统内，也可以单独购买，是一种已知的泛 HDAC 抑制剂，可用作阳性对照抑制剂。曲谷菌素 A 以溶于 DMSO 中的 10mM 的溶液形式提供。

HeLa 细胞核提取物（ HeLa Nuclear Extract ），包含在 HDAC-Glo^™ I/II 筛选系统内，也可以单独购买，可作为组蛋白去乙酰酶的活性来源。稀释的提取物也可用作 HDAC-Glo^™I/II 检测试剂盒的对照。

HDAC-Glo^™ I/II 对照底物只能单独购买，是 HDAC-Glo^™ I/II 底物的非乙酰化形式，两者具有相同的氨基酸序列。 HDAC-Glo^™ I/II 对照底物可与 HDAC-Glo^™ I/II 检测试剂盒及筛选系统一起使用，用于验证二级筛选中真实的 HDAC 抑制作用。对照底物以 10mM 的浓度提供；当与 HDAC-Glo^™ I/II 检测试剂盒或筛选系统中的 HDAC-Glo^™ 试剂一起使用时，在 96 孔板检测模式下，足够进行 480 次检测。

特点

• 去乙酰化活性的简单检测方法： 采用单试剂均质检测方法，操作步骤仅需加入 - 混合 - 检测，轻松用于从普通实验到筛选实验的各种实验。

• 灵敏度高： 比荧光法灵敏度高 10-100 倍。

• 快速获得数据： 仅需 15 分钟即可产生最大信号，辉光型信号持久稳定，使得该方法适用于高通量的自动化操作以及不带自动进样器的发光检测仪。

• 灵活选择样品类型： 样品可来源于活细胞、细胞提取物或纯化的重组酶类。

储存条件

将HDAC-Glo™ Assay 的组分与HDAC-Glo™ I/II Control Substrate( 需单独购买) 储存于-20℃。将HeLa Nuclear Extract 储存于-70℃。

图片

（点击放大图片）

HDAC-Glo^™ I/II 检测试剂盒的化学原理。

加入单试剂后， HDAC 的活性将 HDAC-Glo^™ I/II 发光底物去乙酰化，使得底物肽对 HDAC-Glo^™ 试剂介导的特异的蛋白水解切割反应敏感，从而释放氨基萤光素。释放后的氨基萤光素用 Ultra-Glo^™萤火虫萤光素酶反应来测量，产生稳定持久的光信号。去乙酰化、蛋白水解及萤光素酶反应是三个均质的偶联反应，在加入单试剂后几乎同时发生，并在 15–45 分钟后达到稳定状态。

原厂资料：

The HDAC-Glo™ I/II Assays and Screening Systems are single-reagent-addition, homogeneous, luminescent assays that measure the relative activity of histone deacetylase (HDAC) class I and II enzymes from cells, extracts or purified enzyme sources. The assays use an acetylated, live-cell-permeant, luminogenic peptide substrate that can be deacetylated by HDAC activities. Deacetylation of the peptide aminoluciferin substrate is measured using a coupled enzymatic system in which a protease in the Developer Reagent cleaves the deacetylated peptide from the aminoluciferin substrate, releasing aminoluciferin, which is quantified in a reaction using Ultra-Glo™ recombinant firefly luciferase. The assay reaction is typically complete within 15–45 minutes with no sample manipulation. The HDAC-mediated luminescent signal is persistent, with a half-life of greater than 3 hours, allowing batch processing of multiwell plates. The HDAC assay is broadly useful for class I and II enzymes.

The Trichostatin A, included in the HDAC-Glo™ I/II Screening Systems or available separately, is a known pan HDAC inhibitor that may be used as a positive control inhibitor. The Trichostatin A is supplied at a concentration of 10mM in DMSO.

The HeLa Nuclear Extract, included in the HDAC-Glo™ I/II Screening Systems or available separately, may be used as a source of histone deacetylase activity. The diluted extract also can be used as an HDAC-Glo™ I/II Assay chemistry control.

The HDAC-Glo™ I/II Control Substrate, only available separately, is a non-acetylated form of the HDAC-Glo™ I/II Substrate with the same amino acid sequence and can be used with the HDAC-Glo™ I/II Assays and Screening Systems to confirm true HDAC inhibition in secondary screens. The Control Substrate is supplied at a concentration of 10mM and is sufficient for 480 assays in 96-well plate format when combined with the HDAC-Glo™ Reagent prepared with components in the HDAC-Glo™ I/II Assays or Screening Systems.

Features - Benefits

Simple Measurement of Deacetylating Activities: Use a single-reagent-addition, homogeneous, add-mix-measure protocol for easy implementation from benchtop to screening.
Highly Sensitive: Obtain 10- to 100-fold higher sensitivity than comparable fluorescence methods.
Fast Data Acquisition: Achieve maximum signal in as little as 15 minutes with persistent glow-type steady-state signal, making the protocol amenable to automation in high-throughput formats and compatible with luminometers without injectors.
Flexible to Sample Type: Use with viable cells, extracts or purified recombinant enzyme sources.

Applications

Determine HDAC inhibitor potency using purified enzymes, extracts or cells directly in culture plates.
Profile HDAC inhibitors with purified enzymes.
Correlate HDAC inhibitor potency with cellular fate in same-well multiplexed viability assays.
Confirm deacetylase inhibition with control substrates.

注意事项：

G6420 is sufficient for 100 assays at 100μl/assay in a 96-well plate or 400 assays at 25μl/assay in a 384-well plate. G6421 is sufficient for 500 assays at 100μl/assay in a 96-well plate or 2,000 assays at 25μl/assay in a 384-well plate. G6422 is sufficient for 1,000 assays at 100μl/assay in a 96-well plate or 4,000 assays at 25μl/assay in a 384-well plate.

G6430 is sufficient for 100 assays at 100μl/assay in a 96-well plate or 400 assays at 25μl/assay in a 384-well plate. G6431 is sufficient for 500 assays at 100μl/assay in a 96-well plate or 2,000 assays at 25μl/assay in a 384-well plate.

G6550 is supplied at a concentration of 10mM and is sufficient for 480 assays in 96-well plates when combined with the HDAC-Glo™ I/II Reagent.