| 货号 | 95176S |
| 应用 | MeDIP |
| 目标/特异性 | The SimpleDIP™ Hydroxymethylated DNA IP (hMedIP) Kit can be utilized to detect endogenous levels of 5-hydroxymethylcytosine modifications in mammalian cells (see Figure 1). The 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb has been validated for specificity using ELISA, dot blot and hMeDIP assays and shows high specificity for its target DNA modification (see Figures 2-4). A positive control IP spike-in DNA fragment containing 5-hydroxymethylcytosine and positive control primer set for amplification of this fragment are included in the kit. This spike-in DNA and primer set can be used as a positive control for IP with any mammalian cell type. |
| 供应商 | CST |
| 背景 | DNA immunoprecipitation (DIP) is a technique that uses antibodies to immunoenrich for regions of the genome containing modified nucleotides. This assay was first used with a 5-methylcytosine antibody to identify differentially methylated sites within normal and transformed cells (1). Investigators can use the DIP assay to look at specific genomic loci or look across the entire genome by utilizing next-generation sequencing (NGS) (2). When performing the DIP assay, cells are first lysed and the nucleic acids are recovered using phenol-chloroform extraction and ethanol precipitation. RNA is then removed by RNase A digestion, and genomic DNA is isolated by a second round of phenol-chloroform extraction and ethanol precipitation. The resulting genomic DNA is then fragmented by either restriction enzyme digestion or sonication and subjected to immunoprecipitation (IP) using antibodies specific to the modified nucleotide. Any sequences containing the modified nucleotide will be enriched by the immunoselection process. After IP, the DNA is purified and Quantitative Real-Time PCR can be used to measure the amount of enrichment of a particular DNA sequence. Alternatively, the DIP assay can be combined with NGS to provide genome-wide analysis of a specific DNA modification. |
| 存放说明 | 4C and -20C |
| 参考文献 | Weber, M. et al. (2005) Nat Genet 37, 853-62. |
FIGURE 2. 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb #51660 specificity was determined by dot blot. The same sequence of a 387 base pair DNA fragment was generated by PCR using exclusively unmodified cytosine, 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-carboxylcytosine (5-caC), or 5-formylcytosine (5-fC). The respective DNA fragments were blotted onto a nylon membrane, UV cross-linked, and probed with 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb. The top panel shows the antibody only binding to the DNA fragment containing 5-hmC, while the bottom panel shows the membrane stained with methylene blue. | |
Figure 4. The specificity of 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb #51660 was determined by MeDIP. DNA IPs were performed with genomic DNA prepared from mouse embryonic stem cells, spiked with DNA containing either unmodified cytosine, 5-methylcytosine (5-mC), or 5-hydroxymethylcytosine (5-hmC). IPs were performed using SimpleDIP™ Hydroxymethylated DNA IP (hMeDIP) Kit #95176. The enriched DNA was quantified by real-time PCR using primers specific to the spiked-in control DNA sequence. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input DNA, which is equivalent to one. | |
Figure 3. The specificity of 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb #51660 was determined by an ELISA. The antibody was titrated against a single-stranded DNA oligo containing either unmodified cytosine or differentially modified cytosine (5-mC, 5-hmC, 5-caC, or 5-fC). As shown in the graph, the antibody only binds to the oligo containing 5-hmC. | |
FIGURE 1. DNA immunoprecipitations were performed with 1 μg of genomic DNA from mES cells and either 10 μl of 5-Hydroxymethylcytosine (5-hmc) (HMC31) Mouse mAb #51660 or 10 μl of Mouse (G3A1) mAb IgG1 Isotype Control (DIP Formulated) #98528 using SimpleDIP™ Hydroxymethylated DNA IP (hMeDIP) Kit #95176. The enriched DNA was quantified by real-time PCR using mouse Aqp2 exon 1 primers, SimpleDIP™ Mouse Intracisternal-A Particle (IAP) LTR Primers #74803, mouse Lamc3 exon 1 primers, and SimpleChIP® Mouse GAPDH Intron 2 Primers #8986. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input DNA, which is equivalent to one. |
