Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

SimpleDIP™ Methylated DNA IP (MeDIP) Kit #76853

5-mC   5-methylcytosine   5mc   DIP   DIP kit   DNA IP   DNA methylation   DNA-IP   MeDIP   MeDIPkit   methyl-DNA enrichment   Methyl-DNA IP   methylated DNA   methylated DNA enrichment   Methylated DNA IP   methylcytosine  

REACTIVITY

No. Size Price
76853S 1 Kit ( 10 immunoprecipitations ) ¥4,264.00 现货查询 购买询价
Kit Includes Quantity Applications Reactivity Homology† MW (kDa) Isotype
DNA Binding Buffer 12 ml
DNA Wash Buffer (add 4x volume ethanol before use) 2.5 ml
DNA Elution Buffer 1 ml
DNA Purification Columns and Collection Tubes 15 Pack
Proteinase K 20 µl
5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692 100 µl Rabbit IgG
SimpleDIP™ Cell Lysis Buffer 6 ml
SimpleDIP™ DNA-IP Buffer (10X) 5 ml
SimpleDIP™ Human Testis-Specific H2B Promoter Primers 150 µl
ChIP Elution Buffer (2X) 1.5 ml
RNAse A (10 mg/ml) 50 µl
TE Buffer 10 ml
SimpleDIP™ Mouse Intracisternal A-Particle LTR Primers 150 µl
Rabbit (DA1E) mAb IgG XP® Isotype Control (DIP Formulated) 100 µl Rabbit
3 M Sodium Acetate, pH 5.2 1.2 ml
ChIP-Grade Protein G Magnetic Beads #9006 200 µl

Specificity / Sensitivity

The SimpleDIP™ Methylated DNA IP (MeDIP) Kit can be utilized to detect endogenous levels of 5-methylcytosine modifications in mammalian cells (see Figures 1 and 2). The 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb has been validated for specificity using ELISA, dot blot, and MeDIP assays and shows high specificity for its target DNA modification (see Figures 3-5). Positive control primer sets are included for human testis-specific H2B (TSH2B) promoter and mouse intracisternal A-particle (IAP) LTR, two genomic loci known to be methylated in most cell types. The use of other species with the kit requires the design of additional control primer sets.

Description

The SimpleDIP™ Methylated DNA IP (MeDIP) Kit provides enough reagents to perform up to 10 genomic DNA preparations and 10 IPs from mammalian cells and is optimized for 1 μg of genomic DNA per IP. The SimpleDIP™ protocol can be performed in as little as two days and can easily be scaled up or down for use with more or less cells. Cells are first lysed and genomic DNA is extracted and sonicated into small fragments (200-500 bp). DNA IPs are performed using 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb and ChIP-Grade Protein G Magnetic Beads. After elution from the beads, the DNA is purified using DNA purification spin columns provided in the kit. The enrichment of particular DNA sequences can be analyzed by a variety of methods including standard PCR, quantitative real-time PCR, or next-generation sequencing. The SimpleDIP™ 5-Methylcytosine DNA IP Kit provides a highly validated 5-mC monoclonal antibody to ensure specific and robust signal. The kit also contains human and mouse control primer sets to regions of the genome that contain 5-methylcytosine. Thus, the IP of genomic DNA with 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb will enrich for the sequences amplified by the control primer sets, while the IP with Rabbit (DA1E) mAb XP® Isotype Control (DIP Formulated) will not result in any enrichment.

MeDIP

MeDIP

FIGURE 5. The specificity of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692 was determined by MeDIP. DNA IPs were performed with genomic DNA prepared from mouse embryonic stem cells, spiked with control DNA containing either unmodified cytosine, 5-methylcytosine (5-mC), or 5-hydroxymethylcytosine (5-hmc). IPs were performed using 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb and the SimpleDIP™ Methylated DNA IP (MeDIP) Kit #76853. 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.

Dot Blot-DNA

Dot Blot-DNA

FIGURE 3. The specificity of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692 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-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb. The top panel shows the antibody only binding to the DNA fragment containing 5-mC, while the bottom panel shows the membrane stained with methylene blue.

ELISA-DNA Oligo

ELISA-DNA Oligo

FIGURE 4. The specificity of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692 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, 5-fC). As shown in the graph, the antibody only binds to the oligo containing 5-mC.

MeDIP

MeDIP

FIGURE 2. DNA immunoprecipitations were performed with 1 μg of genomic DNA from mES cells and either 10 μl of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692 or 10 μl of Rabbit (DA1E) mAb IgG XP® Isotype Control (DIP Formulated) #75708 using SimpleDIP™ Methylated DNA IP (MeDIP) Kit #76853. The enriched DNA was quantified by real-time PCR using mouse Aqp2 exon 1 primers, SimpleDIP™ Mouse Intracisternal A-Particle 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.

MeDIP

MeDIP

FIGURE 1. DNA immunoprecipitations were performed with 1 μg of genomic DNA from NCCIT cells and either 10 μl of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692 or 10 μl of Rabbit (DA1E) mAb IgG XP® Isotype Control (DIP Formulated) #75708 using SimpleDIP™ Methylated DNA IP (MeDIP) Kit #76853. The enriched DNA was quantified by real-time PCR using human Aqp2 intron 5 primers, human TIMP3 promoter primers, SimpleDIP™ Human Testis-Specific H2B Promoter Primers #65822, and SimpleChIP® Human GAPDH Exon 1 Primers #5516. 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.

Background

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.

  1. Weber, M. et al. (2005) Nat Genet 37, 853-62.
  2. Down, T.A. et al. (2008) Nat Biotechnol 26, 779-85.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

XP is a registered trademark of Cell Signaling Technology, Inc.

SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.

SimpleDIP is a trademark of Cell Signaling Technology, Inc.

Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.

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