Methylated DNA Immunopreciptation (MeDIP) Kit
Antibody-based enrichment of 5-methylcytosine (5-mC) in DNA
MeDIP Kit Overview
The MeDIP Kit is designed to immunoprecipitate and enrich single-stranded DNA (ssDNA) fragments containing 5-methylcytosine (5-mC). The MeDIP Kit contains a monoclonal 5-methylcytosine antibody, a bridging antibody, and the necessary buffers to perform methylated DNA immunoprecipitation. The high specificity and affinity of the antibody enables separation of 5-mC DNA from DNA containing 5-hmC and other DNA methylation variants using single-stranded input DNA.
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 MseI-digested human genomic DNA 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 methylated DNA.
5-Methylcytosine (5-mC) Methylated DNA Immunoprecipitation
Methylated DNA Immunoprecipitation is an immunocapture technique in which an antibody specific for methylated cytosines is used to immunoprecipitate methylated genomic DNA fragments. MeDIP uses single-stranded DNA that has been fragmented by sonication or enzymatic digestion. The addition of a monoclonal antibody specific for 5-methylcytosine in the presence of a bridging antibody enables specific capture of the DNA fragments containing 5-mC methylation. The use of protein G magnetic beads provides quick and efficient capture. Methylated fragments are then eluted from the magnetic beads, resulting in DNA samples that are specifically enriched for 5-mC DNA methylation. 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-mC and 5-hmC.
The high affinity of the 5-mC antibody used in the MeDIP Kit enables detection of methylated 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 requires CpG methylation for proper detection.
MeDIP Kit Advantages
- Specific antibody only enriches 5-mC methylated DNA
- Includes bridging antibody to increase efficiency of DNA capture
- One-step IP reaction
- No need to pre-wash magnetic beads
- Optimized magnetic protocol to minimize hands-on time
- Kit includes positive control human genomic DNA and PCR primers to verify efficiency
Flow Chart of MeDIP Method
Preparation of MeDIP-Enriched Samples for Next-Gen Sequencing (NGS)
Since the MeDIP Kit uses a 5-methylcytosine antibody that only binds methylated cytosines in the context of single-stranded DNA to enrich for methylated genomic DNA, the DNA must be denatured prior to immunoprecipitation. As a result of denaturation, the enriched DNA can not be processed for next-generation sequencing using some common NGS library generation protocols that include adapter ligation to double-stranded DNA.
This problem can be circumvented by either addition of the NGS adapters to fragmented genomic DNA prior to performing MeDIP or using a library preparation protocol that is compatible with ssDNA.
If you intend to perform NGS analysis following methylated DNA immunoprecipitation using the Active Motif MeDIP Kit, we suggest using the Accel-NGS® 1S DNA Library Kits from Swift Biosciences and following the manufacturer's instructions.
MeDIP Kit Contents
The MeDIP Kit contains a mouse monoclonal 5-methylcytosine antibody, a bridging antibody, negative control mouse IgG, PIC, Buffer C, Buffer D, Elution Buffer AM2, Neutralization Buffer, sterile water, PCR tubes, protein G magnetic beads, and a bar magnet for the separation and elution of DNA fragments containing 5-mC.
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 positive control DNA (MseI-digested human genomic DNA) and positive control ZC3H13 PCR primers.
Storage conditions range from room temperature to -20°C; please refer to the manual for proper storage temperatures of each component.
MeDIP Kit Data
The MeDIP Kit is able to selectively enrich for DNA fragments containing 5-methylcytosine DNA methylation from the rest of the genomic DNA population. The MeDIP Kit was tested using 500 ng of the included MseI-digested human genomic DNA in the presence or absence of bridging antibody. 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 ZC3H13 PCR primer set. Results shown in figure 2 demonstrate the importance of using a bridging antibody within the assay. In figure 3, enriched DNA was tested using either the included positive control ZC3H13 PCR primer mix or a negative control GAPDH PCR primer mix.
MeDIP Kit Publications
The following list represents selected publications in which Active Motif's assay has been used:
“Zac1 regulates astroglial differentiation of neural stem cells through Socs3.” by Schmidt-Edelkraut U. et al. (2013) Stem Cells, doi: 10.1002/stem.1405.
“Activation of BDNF signaling prevents the return of fear in female mice.” by Baker-Andresen D. et al. (2013) Learn Mem, 20(5): 237-240.
MeDIP Kit Documents
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