ChIP-IT High Sensitivity®

针对有限样本材料和低丰度靶蛋白的ChIP

 

ChIP-IT High Sensitivity® 概览

ChIP-IT High-Sensitivity®(HS)试剂盒是市场上最灵敏的ChIP试剂盒。它的设计能够进行高质量的DNA富集。对于高丰富的目标蛋白每个ChIP反应可以使用低至1000个细胞。试剂盒也经过优化针对低丰度蛋白质的抗体执行染色质免疫沉淀时提供优异的结果,如转录因子或具有次优结合亲和力的抗体。要了解这些功能与我们提供的其他ChIP试剂盒的差异,请参阅我们的ChIP试剂盒选择指南

更高的信号峰和较低的非特异性DNA背景是通过特制的蛋白G琼脂糖珠和优化的固定,结合和洗涤缓冲液方案来实现的。此外,清洗步骤是通过重力过滤实现的。它比磁珠捕获更快、更容易、更一致。ChIP-IT High-Sensitivity®(HS)试剂盒也经过验证可用于ChIP-Seq,包括使用针对低丰度转录因子的抗体。

ChIP-IT HS
ChIP-IT High Sensitivity (HS) 试剂盒(点击放大)

ChIP-IT High Sensitivity®优势

  • 低丰度转录因子或低结合亲和力抗体的理想选择
  • 对于高丰度目标蛋白,每次免疫沉淀反应仅从1000个细胞中灵敏富集DNA;对于低丰度转录因子,可从50000个细胞中灵敏富集DNA
  • 优化的试剂组合降低了非特异性结合事件引起的背景水平
  • 基于重力过滤的洗涤方案,相比磁珠捕获,更快、更简单、更具一致性
  • 在qPCR和ChIP-Seq分析中,高效的实验流程已在多种样本类型中得到验证

ChIP-IT High Sensitivity®试剂盒是怎么工作的?

Diagram demonstrating the method of the ChIP-IT High Sensitivity Kit from Active Motif
图1:ChIP-IT High Sensitivity染色质免疫沉淀示意图

在ChIP-IT High-Sensitivity®的实验流程中,完整的细胞用一种特殊配方的甲醛缓冲液固定。这种缓冲液可以交联并保持蛋白质/DNA的相互作用。然后用超声将DNA打断成小片段,并与感兴趣的DNA结合蛋白抗体一起孵育。抗体结合的蛋白质/DNA复合物通过使用蛋白G琼脂糖珠进行免疫沉淀,并通过重力过滤洗涤。免疫沉淀后,DNA解交联,蛋白质被蛋白酶K去除,DNA被回收和纯化。ChIP富集的DNA可用于基因特异性分析或全基因组分析。

Active Motif也提供与ChIP-IT High Sensitivity试剂盒配套的产品:

此外,我们的表观遗传学服务可以为您提供ChIP、ChIP-Seq和许多其他基因组范围的数据生成和分析服务。

Become a ChIP Assay Expert
 

ChIP-IT High Sensitivity® 组分 & 储存

请注意,ChIP-IT High Sensitivity试剂盒High Sensitivity Chromatin制备试剂盒是通过干冰装运的,里面含有多种储存温度的试剂。请将每种试剂存放在以下指示的温度。所有试剂均保证在收到之日起6个月内稳定。收到蛋白G琼脂糖珠后,不要再次冷冻。

High Sensitivity Chromatin制备试剂盒是我们的ChIP-IT High Sensitivity试剂盒的配套模块之一,只包含那些在进行全步骤ChIP反应之前进行固定、剪切和裂解条件优化所需的试剂。

ChIP-IT High Sensitivity试剂盒

  • RNase A (10 µg/µl); 储存于 -20°C
  • Proteinase K (10 µg/µl); 储存于 -20°C
  • 100 mM PMSF; 储存于 -20°C
  • Protease Inhibitor Cocktail (PIC); 储存于 -20°C
  • Precipitation Buffer; 储存于 -20°C
  • Carrier; 储存于 -20°C
  • 10X PBS; 储存于 -20°C
  • Blocker; 储存于 -20°C
  • Fixation Buffer; 储存于 4°C
  • Protein G Agarose beads; Store at 4°C
  • Stop Solution; 储存于 室温
  • Chromatin Prep Buffer; 储存于 室温
  • ChIP Buffer; 储存于 室温
  • Wash Buffer AM1; 储存于 室温
  • Elution Buffer AM4; 储存于 室温
  • ChIP Filtration Columns; 储存于 室温
  • 5 M NaCl; 储存于 室温
  • TE, pH 8.0; 储存于 室温
  • Detergent; 储存于 室温
  • DNA Purification Binding Buffer; 储存于 室温
  • 3 M Sodium Acetate; 储存于 室温
  • DNA Purification Wash Buffer; 储存于 室温
  • DNA Purification Elution Buffer; 储存于 室温
  • DNA Purification Columns; 储存于 室温

High Sensitivity Chromatin Prep试剂盒

  • RNase A (10 µg/µl); 储存于-20°C
  • Proteinase K (10 µg/µl); 储存于-20°C
  • 100 mM PMSF; 储存于-20°C
  • Protease Inhibitor Cocktail (PIC); 储存于-20°C
  • Precipitation Buffer; 储存于-20°C
  • Carrier; 储存于-20°C
  • 10X PBS; 储存于-20°C
  • Fixation Buffer; 储存于 4°C
  • Swelling Buffer; 储存于 4°C
  • Stop Solution; 储存于室温
  • Chromatin Prep Buffer; 储存于室温
  • ChIP Buffer; 储存于室温
  • DNA Purification Elution Buffer; 储存于室温
  • 5 M NaCl; 储存于室温
  • TE, pH 8.0; 储存于室温
  • Detergent; 储存于室温
 

ChIP-IT High Sensitivity®数据

检测低丰度蛋白靶点

ChIP-IT High-Sensitivity®试剂盒非常适合与其他ChIP方法检测不到信号的具有挑战性的抗体一起使用,因为该方法的灵敏度足以检测低丰度蛋白质的特定结合。以下是ChIP-IT High-Sensitivity®试剂盒与其他商用ChIP试剂盒的比较。使用未经处理的MCF-7染色质和结合丰度低的转录辅因子NCOA2抗体进行ChIP检测。同时用阴性对照IgG抗体和阴性对照引物组(Active Motif,目录号71001)检测非特异性结合。只有ChIP-IT High-Sensitivity方法能够检测到NCOA2在特定基因组位置的结合(ESRRA启动子),信号比阴性对照高20倍。尽管ESRRA使用竞争对手D的试剂盒检测到qPCR信号,但由于阴性对照引物组的高背景和IgG检测到的非特异性结合,表明其富集水平不够理想。

PCR analysis showing specific enrichment from low abundance target proteins
图2:针对低丰度转录因子的ChIP试剂盒比较

MCF-7染色质是根据每家试剂盒生产厂家的建议从1.5 x 106个细胞中制备的。使用针对低丰度NCOA2蛋白的抗体和阴性对照IgG进行ChIP ,按照试剂盒制造商建议的最佳染色质数量进行实验。富集后,使用ChIP-IT qPCR分析试剂盒(目录号53029)进行qPCR,以使数据标准化并可以直接比较结果。数据表示为每1000个细胞检测到的结合事件。数值为根据反应中染色质数量、最终重悬体积和引物效率调整后的3次重复实验原始数据的平均值。


获得更好的灵敏度

当使用高质量H3K4me3抗体(目录号39915)时,将ChIP-IT High Sensitivity与其他商用ChIP试剂盒进行比较,结果展示的是分别使用ChIP-IT HS、竞争对手M、竞争对手I和竞争对手D的试剂盒进行阳性对照GAPDH启动子(目录号71006)检测的特定信号。由于优化的条件使ChIP-IT High Sensitivity具有高灵敏度,可检测的富集度比竞争对手M和竞争对手I获得的富集度高2-3倍。只有竞争对手D没有表现出良好的富集度,因为从阴性对照qPCR引物组的信号和IgG反应中的高信号来看,非特异性结合很高。

PCR analysis showing specific enrichment from low abundance target proteins
图3:ChIP-IT High Sensitivity显示出比竞争对手ChIP试剂盒更好的富集

MCF-7染色质是根据每家试剂盒生产厂家的建议从1.5 x 106个细胞中制备的。使用H3K4me3(目录号39915)抗体和阴性对照IgG进行 ChIP,按照试剂盒制造商建议的最佳染色质数量进行实验。富集后,使用ChIP-IT qPCR分析试剂盒(目录号53029)进行qPCR,以使数据标准化并可以直接比较结果。数据表示为每1000个细胞检测到的结合事件。数值为根据反应中染色质数量、最终重悬体积和引物效率调整后的3次重复实验原始数据的平均值。


使用更少的样本量

ChIP-IT High Sensitivity试剂盒中包含的经过优化的ChIP缓冲液减少了非特异性DNA的结合,从而使背景水平最小化。这样可以提高灵敏度和富集效率。低背景使得使用ChIP-IT High Sensitivity试剂盒在样本数量较少条件下的应用成为可能。对于高丰度蛋白质,每个免疫沉淀反应只需1000个细胞;对于低丰度蛋白质,每个免疫沉淀反应只需50000个细胞。在下面的实验中,使用组蛋白H3K4me3抗体和从1000至100万个细胞制备的不同数量染色质进行ChIP实验。当仅使用1000个细胞时,阳性对照GAPDH启动子仍能检测到显著的富集。

PCR analysis showing successful ChIP using samples from 1,000 cells
图4:ChIP-IT High Sensitivity适用于有限的样本材料

MCF-7染色质是根据ChIP-IT High Sensitivity试剂盒的说明书从10万到400万个细胞数量范围制备。然后使用Active Motif组蛋白H3K4me3抗体(目录号39915)和阴性对照IgG对指定数量的细胞进行免疫沉淀。富集后,使用ChIP-IT qPCR分析试剂盒(目录号53029)进行qPCR。结果显示使用ChIP-IT High Sensitivity试剂盒,阳性对照GAPDH启动子可从1000个细胞中进行强富集,而阴性对照UNR12引物组则没有富集。数据表示为每1000个细胞检测到的结合事件。数值为根据反应中染色质数量、最终重悬体积和引物效率调整后的3次重复实验原始数据的平均值。


富集的DNA经过ChIP-Seq验证

使用ChIP-IT High Sensitivity试剂盒获得的高质量ChIP DNA,可用于ChIP-Seq等下游应用。Active Motif已经验证了ChIP-IT High Sensitivity试剂盒可以用于针对具有挑战性的低丰度蛋白质和高丰度组蛋白抗体的ChIP-Seq,如下所示。

ChIP-Seq data for the low abundance target NCOA
图5:低丰度转录因子NCOA2的ChIP-Seq数据

使用ChIP-IT High Sensitivity试剂盒对从300万MCF-7细胞制备染色质和低丰度转录辅因子NCOA2蛋白抗体进行ChIP。ChIP DNA经过建库,并使用Illumina®下一代测序平台进行测序后,生成3000万条序列。对序列数据进行分析,以确定人类基因组中所有的NCOA2结合位点。左图显示了11号染色体上100万个碱基对区域的多个结合位点。右图放大显示两个基因启动子的NCOA2信号峰。

ChIP-Seq data using Histone H3K4me3
图6:组蛋白H3K4me3的ChIP-Seq数据

使用ChIP-IT High Sensitivity试剂盒对从100万MCF-7细胞制备的染色质和组蛋白H3K4me3(目录号39915)抗体进行ChIP。ChIP DNA经过建库,并使用Illumina®下一代测序平台进行测序后,生成2400万条序列。对测序数据进行分析,以确定人类基因组中所有的H3K4me3结合位点。左图显示在11号染色体175000bp区域的5个基因的启动子中H3K4me3的存在。右图放大显示单个基因的启动子结合。PPFIA1基因上的结合信号在转录起始位点出现了共同的双峰。

 

ChIP-IT High Sensitivity®常见问题

染色质制备所需的最小细胞数是多少?

ChIP-IT High Sensitivity试剂盒至少需要100000个细胞用于染色质制备,但这些染色质可以用于多个ChIP反应。

我可以用于免疫沉淀的最小和最大染色质量是多少?

对于高丰度组蛋白标记的抗体,由于试剂盒的高灵敏富集,每个ChIP最少仅需1000个细胞(6.7ng染色质)中获得的染色质。对于转录因子的抗体,我们建议每个ChIP使用50000个细胞(330ng)获得的染色质或更多。

对于染色质使用上限,我们建议使用不超过30𝝻g染色质,大致相当于450万个细胞获得的量(粗略估计150万个细胞产生10𝝻g染色质)。该试剂盒的理想染色质范围为10-30𝝻g,即150-450万个细胞的染色质量。

如果我使用悬浮细胞,我应该如何固定细胞?

方案与贴壁细胞相似,首先直接向培养基中的细胞添加1/10体积的完整细胞固定液。

实验过程中哪些步骤可以暂停?

反应可以在以下时间和温度下暂停:

  1. 甲醛固定和离心(完整细胞颗粒)后,-80°C。
  2. 染色质剪切后,-80°C。
  3. DNA回收后,-20°C。

如何打断染色质?

ChIP-IT High Sensitivity试剂盒可与我们的EpiShear™超声仪配合使用。对于其他超声仪,比如Active Motif的PIXUL™ 多样品超声仪或Diagenode和Covaris超声仪,剪切条件会有所不同。请参阅技术说明:ChIP的超声建议或联系技术支持以获取进一步的指导。

我在Bioanalyzer上检测了ChIP-IT High Sensitivity试剂盒打断的DNA,结果似乎存在高分子量DNA。是超声没有起作用吗?

超声可能工作正常,但ChIP-IT High Sensitivity缓冲液可能会导致在Bioanalyzer上出现大分子量条带的假象。我们建议使用琼脂糖凝胶分析染色质打断效率。注意,试剂盒说明书对在琼脂糖凝胶跑胶之前进行高盐、高温变性样本有具体建议。这对于正确的展示片段大小至关重要。

我可以使用哪个抗体?

使用ChIP验证的抗体很重要,因为免疫沉淀中使用的SDS条件可以减少抗体与蛋白质的结合。许多在其他应用中表现良好的抗体(例如,Western)在ChIP中表现不佳。请参阅我们的ChIP验证抗体列表。如果没有ChIP验证的抗体,我们建议查阅文献中是否有用于交联材料的抗体(例如免疫荧光(IF)或免疫组织化学(IHC))。

对于改善细胞裂解有什么建议吗?

  1. 如果杜恩斯匀浆器不足以有效地裂解细胞,可尝试用28-30口径的针头来裂解细胞。将细胞穿过针头4-5次。
  2. 缩短固定时间。
  3. 对于特别难以裂解的细胞,如PBMC(淋巴细胞、单核细胞和树突状细胞),使用High Sensitivity Chromatin Preparation试剂盒(53046),该试剂盒含有低渗溶胀缓冲液,以便更好地裂解细胞,或者使用ChIP-IT PBMC试剂盒(53042)来获得这些细胞类型的完整ChIP解决方案。

我应该在我的ChIP实验中使用哪些对照?

我们建议使用阳性qPCR 引物和阴性qPCR引物对照。阳性对照qPCR引物扩增的是已知的目标转录因子结合或组蛋白修饰存在的区域,阴性对照qPCR引物扩增的是已知的目标转录因子不结合或组蛋白修饰不存在的区域。

此外,还可以使用阳性和阴性抗体对照。阳性抗体对照是识别与感兴趣位点结合的蛋白抗体。一些常见的阳性抗体对照是组蛋白H3或RNA Pol II。抗体阴性对照是不能识别与感兴趣位点结合的蛋白的抗体。一种常用的阴性对照抗体是小鼠IgG。

对于ChIP-seq,通常使用“input DNA对照”来识别错误的“信号峰”或揭示已被复制的基因组区域。这个对照样品经过固定和超声处理,但没有免疫沉淀。我们建议使用每个ChIP反应的DNA量的1-10%作为input对照。

为了使用方便,Active Motif提供了许多包含对照的产品,以验证您的ChIP实验,包括ChIP-IT对照品试剂盒ChIP-IT qPCR引物组即用染色质ChIP-IT qPCR分析试剂盒

我是第一次使用ChIP-IT High Sensitivity试剂盒。我需要优化固定、剪切和裂解条件。在进行免疫沉淀步骤之前,有没有办法在不使用所有染色质准备试剂的情况下进行一些测试?

为了优化剪切效果,我们提供High Sensitivity Chromatin Prep试剂盒。该试剂盒是ChIP-IT High Sensitivity®试剂盒的配套产品, 包含额外的试剂,专门用于优化样品的固定、剪切和裂解,然后再进行整套ChIP反应。


 

ChIP-IT High Sensitivity®发表文献

以下选择性地列出了一些文献


 

ChIP-IT High Sensitivity®文档

 


 

订购ChIP-IT High Sensitivity®

 



您也可能感兴趣:

 
Name Format Cat No. Price  
ChIP-IT High Sensitivity® 16 rxns 53040 ¥7,150 Buy
High Sensitivity Chromatin Preparation 16 rxns 53046 ¥3,640 Buy
ChIP-IT® Fixation Buffer 3 ml 53038 ¥1,170 Buy
Protein G Agarose Columns 30 rxns 53039 ¥4,750 Buy
5 rxns 53037 ¥1,110 Buy
Protein G Agarose Beads 1.2 ml 37499 ¥2,210 Buy
TE, pH 8.0 35 ml 37515 ¥1,240 Buy
ChIP Buffer 50 ml 37516 ¥1,240 Buy
Blocker 0.1 ml 37498 ¥1,240 Buy

ChIP-IT High Sensitivity® Advantages

  • Ideal for low abundance transcription factors or antibodies with low binding affinities
  • Sensitive enrichment of DNA from as little as 1,000 cells per immunoprecipitation reaction for high abundance target proteins and as little as 50,000 cells for low abundance transcription factors
  • Optimized reagents reduce background levels caused by non-specific binding events
  • Filtration based washes offer a faster, easier solution with better consistency than magnetic capture
  • Highly robust procedure has been validated across multiple sample types with proven performance in both qPCR and ChIP-Seq analysis

How does the ChIP-IT High Sensitivity® Kit work?

Diagram demonstrating the method of the ChIP-IT High Sensitivity Kit from Active Motif
Figure 1: Schematic of chromatin immunoprecipitation using ChIP-IT High Sensitivity.

In ChIP-IT High Sensitivity®, intact cells are fixed with a specially formulated formaldehyde buffer, which cross-links and preserves protein/DNA interactions. DNA is then sheared into small fragments using sonication and incubated with an antibody directed against the DNA-binding protein of interest. The antibody-bound protein/DNA complexes are immunoprecipitated through the use of Protein G agarose beads and washed via gravity filtration. Following immunoprecipitation, the DNA cross-links are reversed, the proteins are removed by Proteinase K and the DNA is recovered and purified. ChIP enriched DNA can be used for either gene-specific or whole-genome analysis.

Detect Low Abundance Protein Targets

The ChIP-IT High Sensitivity® Kit is ideal for use with challenging antibodies that do not give signal with other ChIP methods, as the method is sensitive enough to detect specific binding of even low abundance proteins. Below is a comparison of ChIP-IT High Sensitivity with other commercially available ChIP kits. ChIP was performed using untreated MCF-7 chromatin and an antibody that binds the low abundance nuclear receptor co-activator 2 (NCOA2) transcriptional co-factor. It was also performed with a negative control IgG and a negative control primer set (Active Motif Catalog No. 71001) to monitor non-specific binding. Only the ChIP-IT High Sensitivity method was able to detect NCOA2 binding at specific genomic locations (ESRRA promoter), providing approximately 20-fold higher enrichment than the negative controls. Although ESRRA shows qPCR signals using the kit from Competitor D, the enrichment levels are poor due to the high background from the negative control primer set and the non-specific binding detected with the IgG.

PCR analysis showing specific enrichment from low abundance target proteins
Figure 1: Comparison of ChIP kits targeting a low abundance transcription factor.

MCF-7 chromatin was prepared according to each manufacturer's recommendations for their ChIP assay from 1.5 x 106 cells. ChIP was performed with the optimal amount of chromatin as suggested by the protocol for each manufacturer using an antibody for the low abundance NCOA2 protein and a negative control IgG. Following enrichment, qPCR was performed using the ChIP-IT qPCR Analysis Kit (Catalog No. 53029) in order to normalize the data and allow for direct comparison of the results. Data is expressed as binding events detected per 1,000 cells which represents the average of the raw data triplicates adjusted for the amount of chromatin in the reaction, the resuspension volume and the primer efficiency.


Achieve Greater Sensitivity

A comparison of ChIP-IT High Sensitivity to other commercially available ChIP Kits when using the high-quality H3K4me3 antibody (Catalog No. 39915) shows specific signal for the positive control GAPDH promoter (Catalog No. 71006) using ChIP-IT HS, Competitor M and Competitor I's Kits. Due to the optimized conditions with the ChIP-IT High Sensitivity, the detectable enrichment was 2-3 fold higher than the enrichment obtained from Competitor M and Competitor I. Only Competitor D did not show good enrichments as a result of high non-specific binding as seen with the signal from the negative control qPCR primer set and the high signal in the IgG reactions.

PCR analysis showing specific enrichment from low abundance target proteins
Figure 2: ChIP-IT High Sensitivity shows better enrichment than competitor ChIP Kits.

MCF-7 chromatin was prepared according to each manufacturer's recommendations for their ChIP assay from 1.5 x 106 cells. ChIP was performed with the optimal amount of chromatin as suggested by the protocol for each manufacturer using an antibody for the abundant Histone H3K4me3 (Catalog No. 39915) and a negative control IgG. Following enrichment, qPCR was performed using the ChIP-IT qPCR Analysis Kit (Catalog No. 53029) in order to normalize the data and allow for direct comparison of the results. Data is expressed as binding events detected per 1,000 cells which represents the average of the raw data triplicates adjusted for the amount of chromatin in the reaction, the resuspension volume and the primer efficiency.


Use Less Sample Material

The optimized ChIP buffers included in the ChIP-IT High Sensitivity Kit reduce the presence of non-specific DNA, thereby minimizing background levels. This results in a increased sensitivity and better enrichment. The reduced background makes it possible to use the ChIP-IT High Sensitivity Kit with as little as 1,000 cells per immunoprecipitation reaction for high abundance proteins and as little as 50,000 cells per immunoprecipitation reaction for low abundance proteins. In the experiment below, ChIP was performed using the Histone H3K4me3 antibody and chromatin from 1,000 to 1 million cells. Significant enrichment was still detectable at the positive control GAPDH promoter when using only 1,000 cells.

PCR analysis showing successful ChIP using samples from 1,000 cells
Figure 3: ChIP-IT High Sensitivity is suitable for use with limited sample material.

MCF-7 chromatin was prepared according to the ChIP-IT High Sensitivity manual using between 100,000 to 4 million cells per chromatin preparation. The immunoprecipitation was then performed using the indicated number of cells with Active Motif's Histone H3K4me3 antibody (Catalog No. 39915) and a negative control IgG. Following enrichment, qPCR was performed using the ChIP-IT qPCR Analysis Kit (Catalog No. 53029). The ChIp-IT High Sensitivity Kit shows strong enrichment with the positive control GAPDH promoter from as little as 1,000 cells and no enrichment with the negative control Untr12 primer set. Data is expressed as binding events detected per 1,000 cells which represents the average of the raw data triplicates adjusted for the amount of chromatin in the reaction, the resuspension volume and the primer efficiency.


Enriched DNA is ChIP-Seq Validated

The ChIP DNA obtained from the ChIP-IT High Sensitivity Kit is of high quality and can be used in downstream applications such as ChIP-Seq or ChIP-chip. Active Motif has validated the ChIP-IT High Sensitivity Kit for ChIP-Seq with both challenging antibodies that target low abundance proteins and highly abundant histone proteins as shown below.

ChIP-Seq data for the low abundance target NCOA
Figure 4: ChIP-Seq data for the low abundance transcription factor NCOA2.

ChIP was performed using the ChIP-IT High Sensitivity Kit, an antibody against the low abundance transcriptional co-activator NCOA2 and chromatin from 3 million MCF-7 cells. ChIP DNA was processed and 30 million sequence tags were generated using the Illumina® Next-Gen sequencing platform. The sequence data was analyzed to identify all NCOA2 binding sites in the human genome. The left panel shows multiple binding sites across a 1 million base pair region on chromosome 11. The image in the right panel is zoomed in to show NCOA2 peaks in the promoters of two genes.

ChIP-Seq data using Histone H3K4me3
Figure 5: ChIP-Seq data for Histone H3K4me3.

ChIP was performed using the ChIP-IT High Sensitivity Kit, an antibody against Histone H3K4me3 (Catalog No. 39915) and chromatin from 1 million MCF-7 cells. ChIP data was processed and 24 million sequence tags were generated using the Illumina® Next-Gen sequencing platform. The sequencing data was analyzed to identify all the H3K4me3 binding sites in the human genome. The left panel shows the presence of the H3K4me3 in the promoters of 5 genes across a 175,000 bp region on chromosome 11. The image in the right panel is zoomed in to show binding in the promoter of a single gene. Binding at the PPFIA1 gene shows the common double peak with depletion right at the transcriptional start site.

Contents & Storage

Please note that the ChIP-IT High Sensitivity Kit is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please store each component at the temperature indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Do not re-freeze the Protein G Agarose Beads after you have received this kit. This kit includes the following components:

  • RNase A (10 µg/µl); Store at -20°C
  • Proteinase K (10 µg/µl); Store at -20°C
  • 100 mM PMSF; Store at -20°C
  • Protease Inhibitor Cocktail (PIC); Store at -20°C
  • Precipitation Buffer; Store at -20°C
  • Carrier; Store at -20°C
  • 10X PBS; Store at -20°C
  • Blocker; Store at -20°C
  • Fixation Buffer; Store at 4°C
  • Protein G Agarose beads; Store at 4°C
  • Stop Solution; Store at RT
  • Chromatin Prep Buffer; Store at RT
  • ChIP Buffer; Store at RT
  • Wash Buffer AM1; Store at RT
  • Elution Buffer AM4; Store at RT
  • ChIP Filtration Columns; Store at RT
  • 5 M NaCl; Store at RT
  • TE, pH 8.0; Store at RT
  • Detergent; Store at RT
  • DNA Purification Binding Buffer; Store at RT
  • 3 M Sodium Acetate; Store at RT
  • DNA Purification Wash Buffer; Store at RT
  • DNA Purification Elution Buffer; Store at RT
  • DNA Purification Columns; Store at RT

Please note that the High Sensitivity Chromatin Preparation Kit is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please store each component at the temperature indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Do not re-freeze the Protein G Agarose Beads after you have received this kit. This kit includes the following components:

  • RNase A (10 µg/µl); Store at -20°C
  • Proteinase K (10 µg/µl); Store at -20°C
  • 100 mM PMSF; Store at -20°C
  • Protease Inhibitor Cocktail (PIC); Store at -20°C
  • Precipitation Buffer; Store at -20°C
  • Carrier; Store at -20°C
  • 10X PBS; Store at -20°C
  • Fixation Buffer; Store at 4°C
  • Swelling Buffer; Store at 4°C
  • Stop Solution; Store at RT
  • Chromatin Prep Buffer; Store at RT
  • ChIP Buffer; Store at RT
  • DNA Purification Elution Buffer; Store at RT
  • 5 M NaCl; Store at RT
  • TE, pH 8.0; Store at RT
  • Detergent; Store at RT

Some select publications are listed below.