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EpiQuik Histone Demethylase (H3-K4 Specific) Activity/Inhibition Assay Kit
EpiQuik 组蛋白去甲基酶（H3-K4 特异性）活性/抑制检测试剂盒

Base Catalog # P-3074
基础目录号码 P-3074

PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE
请在使用前阅读完整的用户指南

The EpiQuik Histone Demethylase (H3-K4 Specific) Activity/Inhibition Assay Kit is very suitable for measuring histone demethylase (H3-K4 specific) activity/inhibition from a broad range of species including mammalian cells/tissues, plants, and bacteria.
EpiQuik 组蛋白去甲基酶（H3-K4 特异性）活性/抑制检测试剂盒非常适用于测量来自包括哺乳动物细胞/组织、植物和细菌在内的广泛物种范围内的组蛋白去甲基酶（H3-K4 特异性）活性/抑制。

KIT CONTENTS 套件内容

Components 48 assays 96 assays
P-3074-48 P-3074-96
HD1 (10X Wash Buffer)
HD1（10 倍洗涤缓冲液）
HD2 (HDM Assay Buffer)
HD2（HDM 检测缓冲液）
HD3 (HDM Substrate)* HD3（HDM 基板）*
HD4 (HDM Standard,  HD4（HDM 标准，
HD5 (Capture Antibody,
HD5（捕获抗体，
HD6 (Detection Antibody,
HD6（检测抗体，
HD7 (Fluoro Developer)* HD7（氟开发剂）*
HD8 (Fluoro Enhancer)* HD8（氟增强剂）*
HD9 (Fluoro Dilution) HD9（氟稀释）
8-Well Assay Strip (with Frame)
8 孔分析条（带框）		 6 12
User Guide 1 1
* For maximum recovery of the products, centrifuge the original vial after thawing prior to opening
为了最大限度地恢复产品，解冻前打开原始小瓶后离心
the cap.

SHIPPING & STORAGE 运输和储存

The kit is shipped in two parts: one part at ambient room temperature, and the second part on frozen ice packs at .
该套件分两部分发货：一部分在室温下，另一部分在 冰袋上冷冻。
Upon receipt: (1) Store HD3, HD4, HD6, and HD7 at away from light; (2) Store all other components at away from light. The kit is stable for up to 6 months from the shipment date, when stored properly.
收到后：（1）将 HD3、HD4、HD6 和 HD7 存放在离光 的地方；（2）将所有其他组件存放在离光 的地方。如果妥善存放，该套件从发货日期起稳定期长达 6 个月。
Note: Check if wash buffer, HD1, contains salt precipitates before using. If so, warm (at room temperature or ) and shake the buffer until the salts are re-dissolved.
请注意：在使用洗涤缓冲液 HD1 之前，请检查是否含有盐沉淀。如果有，请加热（室温或 ）并摇动缓冲液直到盐重新溶解。

MATERIALS REQUIRED BUT NOT SUPPLIED
所需材料但未提供

Orbital shaker  轨道摇床
  • Pipettes and pipette tips
    移液管和移液管尖头
  • microcentrifuge tubes
    微离心管

GENERAL PRODUCT INFORMATION
通用产品信息

Usage Limitation: The EpiQuik Histone Demethylase (H3-K4 Specific) Activity/Inhibition Assay Kit is for research use only and is not intended for diagnostic or therapeutic application.
使用限制：EpiQuik 组蛋白去甲基化酶（H3-K4 特异性）活性/抑制检测试剂盒仅用于研究，不适用于诊断或治疗应用。
Quality Control: Epigentek guarantees the performance of all products in the manner described in our product instructions.
质量控制：Epigentek 保证所有产品的性能符合我们产品说明中描述的方式。
Product Updates: Epigentek reserves the right to change or modify any product to enhance its performance and design.
产品更新：Epigentek 保留更改或修改任何产品以增强其性能和设计的权利。
Intellectual Property: The EpiQuik Histone Demethylase (H3-K4 Specific) Activity/Inhibition Assay Kit and methods of use contain proprietary technologies by Epigentek. EpiQui is a trademark of Epigentek Group Inc.
知识产权：EpiQuik 组蛋白去甲基酶（H3-K4 特异性）活性/抑制检测试剂盒及其使用方法包含 Epigentek 的专有技术。EpiQuik 是 Epigentek Group Inc. 的商标。

A BRIEF OVERVIEW 简要概述

Lysine histone methylation is one of the most robust epigenetic marks and is essential for the regulation of multiple cellular processes. The methylation of seems to be of particular significance, as it is associated with active regions of the genome. H3-K4 methylation was considered irreversible until the identification of a large number of histone demethylases indicated that demethylation events play an important role in histone modification dynamics. So far, at least 2 classes of H3-K4 specific histone demethylase, LSD1 and JARIDs, have been identified. LSD1 can remove di- and mono-methylation from by using an amine oxidase reaction, while JARIDs such as RBP2, PLU-1, SMCX, and SMCY catalyzes the removal of methylation by using a hydroxylation reaction and required iron and -ketoglutarate as cofactors. H3-K4 specific demethylases are found to be involved in some pathological processes such as cancer progression. Inhibition of the enzymes may lead to re-methylation of H3-K4 and silencing of H3K4 enriched active genes. There are few methods currently available for measuring activity/inhibition of specific methylases using a variety of cells/tissues.
赖氨酸组蛋白甲基化是最强大的表观遗传标记之一，对于调控多种细胞过程至关重要。 的甲基化似乎具有特殊意义，因为它与基因组的活跃区域相关。直到发现大量组蛋白去甲基化酶，H3-K4 甲基化被认为是不可逆的，这表明去甲基化事件在组蛋白修饰动态中起着重要作用。到目前为止，已经鉴定出至少 2 类 H3-K4 特异性组蛋白去甲基化酶，LSD1 和 JARIDs。LSD1 可以通过胺氧化酶反应从 中去除二甲基化和单甲基化，而 RBP2、PLU-1、SMCX 和 SMCY 等 JARIDs 则通过羟化反应催化去除甲基化，并需要铁和 -酮戊二酸作为辅因子。发现 H3-K4 特异性去甲基化酶参与了一些病理过程，如癌症进展。抑制这些酶可能导致 H3-K4 的重新甲基化和 H3K4 富集活跃基因的沉默。目前很少有方法可用于测量 特异性甲基转移酶的活性/抑制，可使用各种细胞/组织。
The EpiQuik Histone Demethylase (H3-K4 Specific) Activity/Inhibition Assay Kit uses a proprietary and unique procedure to measure activity/inhibition of specific histone demethylases using cell/tissue extracts. This kit has the following features:
EpiQuik 组蛋白去甲基化酶（H3-K4 特异性）活性/抑制检测试剂盒采用专有和独特的程序，用于测量细胞/组织提取物中 特异性组蛋白去甲基化酶的活性/抑制。该试剂盒具有以下特点：
  • Fast procedure, which can be finished within 3 hours.
    快速程序，可在 3 小时内完成。
  • Innovative fluorescent assay without the need for radioactivity, extraction, or chromatography.
    无需放射性、提取或色谱的创新荧光测定法。
  • Direct measurement of HDM (H3-K4 specific) activity and inhibition without interference by thiol-containing chemicals such as DTT, GSH, and 2-mercaptoethanol.
    直接测量 HDM（H3-K4 特异性）活性和抑制，无需受到 DTT、GSH 和 2-巯基乙醇等含硫化学物质的干扰。
  • Strip microplate format makes the assay flexible: manual or high throughput analysis.
    条形微孔板格式使得检测灵活：手动或高通量分析。
  • Simple, reliable, and consistent assay conditions.
    简单、可靠且一致的检测条件。

PRINCIPLE & PROCEDURE 原则与程序

The EpiQuik Histone Demethylase (H3-K4 Specific) Activity/Inhibition Assay Kit is designed for measuring total histone demethylase (H3-K4 specific) activity/inhibition. In the assay with this kit, the unique di-methylated histone substrate is stably captured on the strip wells. Active HDMs bind to and demethylate histone substrate. The remaining un-demethylated substrate can be recognized with a high affinity anti-methylated histone antibody. The ratio or amount of the un-demethylated histone, which is inversely proportional to HDM enzyme activity, can then be fluorometrically quantified.
EpiQuik 组蛋白去甲基酶（H3-K4 特异性）活性/抑制检测试剂盒旨在测量总组蛋白去甲基酶（H3-K4 特异性）活性/抑制。在使用该试剂盒进行检测时，独特的二甲基化组蛋白 底物被稳定地捕获在条形孔中。活性 HDMs 与组蛋白 底物结合并去甲基化。剩余的未去甲基化底物可以通过高亲和力的抗甲基化组蛋白 抗体识别。未去甲基化组蛋白的比例或数量与 HDM 酶活性成反比，然后可以通过荧光定量法定量化。
Schematic Procedure for Using the EpiQuik Histone Demethylase (H3-K4 Specific) Activity/Inhibition Assay Kit
使用 EpiQuik 组蛋白去甲基酶（H3-K4 特异性）活性/抑制检测试剂盒的示意流程

PROTOCOL 协议

  1. Prepare nuclear extracts by using you own successful method. For your convenience and the best results, Epigentek offers a nuclear extraction kit (Cat. No. OP-0002-1) optimized for use in EpiQuik "Th series. Nuclear extracts can be used immediately or stored at for future use.
    使用您自己成功的方法制备核提取物。为了您的方便和最佳结果，Epigentek 提供了一个核提取试剂盒（Cat. No. OP-0002-1），经过优化，可用于 EpiQuik“Th”系列。核提取物可以立即使用或存储在 以备将来使用。
  2. Determine the number of strip wells required. Leave these strips in the plate frame (remaining unused strips can be placed back in the bag. Seal the bag tightly and store at ). Dilute HDI 10X Wash Buffer with distilled water (pH 7.2 to 7.5 ) into a 1 X concentration (IX HDI).
    确定所需的条带孔数量。将这些条带留在板框中（剩余未使用的条带可以放回袋中。密封袋子并存放在 ）。用蒸馏水稀释 HDI 10X 洗涤缓冲液（pH 值为 7.2 至 7.5）至 1 倍浓度（1X HDI）。
  3. Dilute HD3 at a ratio with , and add of the diluted HD3 into each well. For preparation of the standard curve, add of into the wells (no HD3 added), followed by adding of HD4 at different concentrations ( ). Cover the wells with Parafilm M and incubate at room temperature for 30-45 minutes.
    将 HD3 以 比例稀释，然后将稀释后的 HD3 加入每个孔中 。为了准备标准曲线，将 的 加入孔中（不加 HD3），然后加入不同浓度的 HD4（ ） 。用 Parafilm M 盖住孔，室温孵育 30-45 分钟。
  4. Aspirate and wash each well with of 1 X HDI two times.
    用 的 1 倍 HDI 两次冲洗每个孔。
  5. Add of HD2 and of nuclear extracts to each strip well. Mix, cover the strip wells, and incubate at for 60 minutes. For the control, add of HD2 instead of nuclear extract. For HDM inhibition, add l of tested inhibitors at different amounts and reduce HD2 volume to . For the blank, add of HD2 into the blank wells (no HD3 added).
    将每个条带孔中加入 的 HD2 和 的核提取物 。混合，盖好条带孔，孵育 60 分钟。对照组中，加入 的 HD2 代替核提取物。对于 HDM 抑制，加入 升测定抑制剂以不同量，并将 HD2 体积减少至 。对于空白组，将 的 HD2 加入空白孔中（不加 HD3）。
  6. Aspirate and wash each well with of 1 X HD1 three times.
    用 的 1 倍 HD1 冲洗每个孔。
  7. Dilute HD5 (at a ratio) to with 1 X HD1. Add of the diluted HD5 to each strip well and incubate at room temperature for 60 minutes on an orbital shaker (50-100 rpm).
    将 HD5（比例为 ）稀释至 ，使用 1 倍 HD1。向每个条带孔中加入稀释后的 HD5 ，在轨道摇床上室温孵育 60 分钟（50-100 rpm）。
  8. Aspirate and wash each well with of four times.
    用 四次冲洗每个孔。
  9. Dilute HD6 (at a ratio) to with HD1. Add of the diluted HD6 to each strip well and incubate at room temperature for 25-30 min.
    将 HD6（按 比例）稀释至 ，使用 HD1。向每个条形孔中加入稀释后的 HD6 ，在室温下孵育 25-30 分钟。
  10. Aspirate and wash each well with of five to six times.
    用 的 冲洗每个孔五到六次。
  11. Prepare the Fluoro-Development Solution by adding l of HD7 and l of HD8 into each 400 of HD9.
    通过将 升 HD7 和 升 HD8 加入每 400 的 HD9 中来准备氟发展液。
  12. Add of the Fluoro-Development Solution into the wells and incubate at room temperature for 2-5 minutes away from light. Measure and read fluorescence on a fluorescence microplate reader at .
    将 的氟发展液加入孔中，在室温下远离光照孵育 2-5 分钟。在荧光微孔板阅读器上测量和读取 的荧光。
Note: If the strip well frame does not fit the fluorescence reader, transfer the solution to a standard 96 -well microplate and read fluorescence at .
请注意：如果条形框不适合荧光读数器，请将溶液转移到标准 96 孔微孔板中，并在 处读取荧光。
  1. Calculate HDM (H3-K4) activity or inhibition. For simple calculation:
    计算 HDM（H3-K4）的活性或抑制。简单计算：
For an accurate calculation, plot Delta RFU value versus amount of HD4 and determine the slope as delta .
为了准确计算，请绘制 Delta RFU 值与 HD4 量之间的图，并确定斜率为 delta 。
Calculate HDM (H3-K4) activity using the following formula:
使用以下公式计算 HDM（H3-K4）活性：

TROUBLESHOOTING 故障排除

No Signal for the Sample
样本无信号

The protein sample is not properly extracted.
蛋白质样本提取不正确。
The protein amount is added into
蛋白质量已添加到
Ensure the protein extraction protocol is suitable for nuclear protein extraction.
确保蛋白质提取方案适用于核蛋白提取。
Ensure extract contains a sufficient amount of
确保提取物含有足够的
well insufficiently. 很不足。
The sample is not prepared from frozen cells or tissues.
样本不是由冷冻的细胞或组织制备的。
Nuclear extracts are incorrectly stored.
核提取物存储不正确。
Reagents are added incorrectly.
试剂添加错误。
Incubation time and temperature are incorrect.
孵化时间和温度不正确。
Absence of HDM (H3-K4) activity in the sample due to treatment.
由于处理，样本中缺乏 HDM（H3-K4）活性。

High Background Present for the Blank
高背景出现在空白处

The well is not washed sufficiently.
井水没有洗干净。
Overdevelopment. protein.
过度发育。蛋白质。
The nuclear extracts from frozen cells/tissue significantly lose enzyme activity. A fresh sample should be used.
从冷冻细胞/组织中提取的核提取物明显失去酶活性。 应使用新鲜样本。
Ensure the nuclear extracts are stored at .
确保核提取物存放在 。
Check if reagents are added in order and if any steps of the procedure may have been omitted by mistake.
检查试剂是否按顺序添加，以及程序中是否有任何步骤被错误地省略。
Ensure the incubation time and temperature described in the protocol are followed correctly.
确保按照方案中描述的孵育时间和温度正确进行。
N/A.
Check if wash at each step is performed according to the protocol.
检查每一步的洗涤是否按照规程执行。
Decrease development time in step 12.
在第 12 步减少开发时间。
P-3075A
EpiQuik Histone Demethylase LSD1 Inhibitor Screening Assay Core Kit
EpiQuik 组蛋白去甲基酶 LSD1 抑制剂筛选检测核心试剂盒
P-3076 EpiQuik Histone Demethylase LSD 1 Activity/Inhibition Assay Kit
P-3076 EpiQuik 组蛋白去甲基酶 LSD 1 活性/抑制检测试剂盒