羟甲基化免疫沉淀试剂盒 货号：E-55010
hMeDIP
背景资料：The hMeDIP Kit is designed to immunoprecipitate and enrich for DNA fragments containing 5-hydroxymethylcytosine (5-hmC). The hMeDIP Kit contains a highly specific purified 5-hmC antibody and the necessary buffers to perform methylated DNA immunoprecipitation (MeDIP). The affinity of the antibody enables separation of 5-hmC DNA from 5-mC DNA. Additionally, the antibody works to efficiently immunoprecipitate either double-stranded or single-stranded input DNA. For complete details, click the 5-hmC Info tab below..
Active Motif's fast, magnetic protocol has been streamlined to minimize the number of wash and incubation steps, saving you valuable time. The kit also includes unmethylated, 5-methylcytosine and 5-hydroxymethylcytosine DNA controls and PCR primers that can be used to verify the efficiency of the enrichment. Additionally, the kit contains a bar magnet for easy separation and elution of the enriched hydroxymethylated DNA. Click the Data tab below to see results from the kit; manuals can be downloaded under the Documents tab
产品描述：Methylated DNA Immunoprecipitation (MeDIP) is an immunocapture technique in which an antibody specific for methylated cytosines is used to immunoprecipitate methylated genomic DNA fragments. For hMeDIP, the antibody used for immunocapture is a 5-hydroxymethylcytidine antibody that recognizes DNA fragments containing 5-hydroxymethylcytosine (5-hmc). This selectivity is important as most common approaches to analyze DNA methylation, such as enzymatic approaches and bisulfite conversion, are unable to distinguish between 5-hmC and 5-mC..
The 5-hydroxymethylcytosine modification results from the enzymatic conversion of 5-methylcytosine into 5-hydorxymethylcytosine by the TET family of cytosine oxygenases. While the precise function of 5-hmC has yet to be determined, it has been postulated that it could represent a pathway to demethylated DNA, as 5-hydroxymethylcytosine is repaired as mismatched DNA and replaced with unmethylated cytosine.
The affinity of the antibody used in the hMeDIP kit enables detection of hydroxymethylated cytosines regardless of their context. This means that the methylation does not need to occur in the context of a CpG dinucleotide, unlike methyl-binding protein (MBD) affinity enrichment methods which require CpG methylation for proper detection. Another advantage of the hMeDIP Kit is that the 5-hmC antibody works efficiently to immunoprecipitate either double-stranded or single-stranded DNA
产品特点：
1.Specific antibody only enriches 5-hmC methylated DNA；
2.Works with either double-stranded or single-stranded DNA；
3.Optimized magnetic protocol to minimize hands-on time；
4.Kit includes unmethylated, 5-methylcytosine and 5-hydroxymethylcytosine control DNAs and PCR primers to verify efficiency；
产品应用：The hMeDIP Kit is able to selectively enrich for DNA fragments containing 5-hydroxymethylcytosine DNA methylation from the rest of the genomic DNA population. The hMeDIP Kit was tested using the included methylated DNA controls. The control DNAs contain a 338 base pair DNA fragment consisting of 122 cytosine residues that are completely unmethylated, completely 5-methylcytosine methylated or completely 5-hydroxymethylcytosine metylated. These DNA controls were spiked into human, male genomic DNA at a ratio of 1:20,000 (spike:genomic DNA) and run through the hMeDIP Kit. Eluted DNA was purified with Active Motif's Chromatin IP DNA Purification Kit (Catalog No. 58002) and tested in real time PCR with the included PCR primer set. Results are shown in Figure 1 that indicate the specificity of the 5-hmC antibody for detecting only the 5-hydroxymethylated DNA. Fold enrichment was calculated by normalizing the DNA enriched with the 5-hmC antibody against the DNA enriched with the negative control rabbit IgG antibody.
保存建议：The hMeDIP Kit contains a purified 5-hydroxymethylcytidine polyclonal antibody, a negative control rabbit IgG antibody, PIC, Buffer C, Buffer D, Elution Buffer AM2, Neutralization Buffer, PCR tubes, Protein G magnetic beads and a bar magnet for the separation and elution of DNA fragments containing 5-hmC. Protocols are provided for the fragmentation of sample DNA all the way through to real time PCR analysis of a locus of interest..
The kit also includes DNA controls and PCR primers. A 338 bp sequence of the APC locus that is unmethylated, fully hydroxymethylated or fully methylated is included for optional spike experiments to determine the efficiency of the immunoprecipitation. APC PCR primers are included to verify the enrichment of the DNA controls.
Storage condiitons range from room temperature to -20°C; please refer to the manual for proper storage temperatures of each component
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关于5-hmC
5-hmC是近年来在动物组织中发现的，由胞嘧啶修饰而来。5-hmC在表观遗传学上的功能可能与5-甲基化胞嘧啶(5-mC)不同。尽管到现在为止还不确知其功能，有研究者猜测它在调控基因的表达与关闭过程中起着重要的作用。5-mC的发现让我们不得不重新评估DNA甲基信息，也不得不监测人类的健康组织和病理组织之间5-mC相对分布的差异。在EPI公司的MethylFlash技术之前，我们还没有发现任何直接的常规方法来检测5- hmC,以及区分5-hmC和5-mC
5-hmC 和 5-mC的区别
时下常用的DNA甲基化分析方法包括限制内切酶酶切和亚硫酸氢盐或MeDIP介导的MS-PCR和测序，这些技术都不适合用来检测5-hmC，因为它与5-mC事实上很难用这类方法区分开来。为了解决这个问题，EPIk研制了MethylFlash羟甲基化DNA定量试剂盒(荧光法)。本试剂盒提供了一种很经济的方法来检测5-羟甲基化胞嘧啶，并且区分5-hmC, 5-mC, 和 C，使得研究者能够重新评估他们的DNA甲基化信息，也能够在新样品中寻找DNA羟甲基化。
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