DNA methylation is a major epigenetic modification that occurs primarily at the C5 position of the cytosine ring. Methylated DNA immunoprecipitation (MeDIP) is a technique that captures and enriches methylated DNA using 5mC-specific antibodies. This technique enables the isolation of methylated DNA fragments for subsequent analysis.
We present a standard MeDIP method. In this protocol, antibodies against 5mC are used to immunoprecipitate methylated DNA. And we can also provide highly specific antibodies and the necessary solutions and reagents. The rapid and streamlined protocol helps you save valuable time.
|Stages||Solutions and Reagents|
|Sample Preparation||Digestion buffer, nucleic acid extraction solution, DNA purify cation kit, buffer TE, IP buffer|
|Immunoprecipitation||IP buffer, anti-5mC antibody, dilution buffer, digestion buffer, wash buffer, binding buffer, elution buffer|
Collect the cells and add digestion buffer for cell lysis. Then extract the nucleic acids from the sample and perform DNA purification. Before starting the assay, genomic DNA should be fragmented to produce fragments between 200 and 600 base pairs in size. Transfer the quantitative genomic DNA sample into a microcentrifuge tube and adjust the final volume. Perform sonication on ice using a sonicator. If desired, the sonicated DNA can be verified with agar gel analysis.
To further increase the binding affinity of the antibody, the DNA fragment is denatured to produce single-stranded DNA. After denaturation, place the DNA on ice immediately and then add IP buffer to mix thoroughly. Then incubate the DNA with the appropriate antibody dilution.
Add the methylated DNA/antibody complex to the beads and incubate. At the end of the incubation, centrifuge the bead-bound methylated DNA/antibody complex and discard the supernatant. Add IP buffer and wash several times. If using magnetic beads, no centrifugation is required after incubation with the DNA/antibody complex. Collect the beads directly using the magnetic rack and repeat the wash.
Resuspend the collected magnetic beads in the elution buffer and heat to incubate. After incubation is complete, centrifuge the supernatant and repeat the elution step several times, combining the eluates in a single tube. If using magnetic beads, place the suspension on a magnetic separation holder and repeat the elution step as above to collect the supernatant.
After MeDIP, DNA methylation can be analyzed using a variety of downstream applications. To ensure that your enriched mDNA samples are rich and accurate, you can refer to our troubleshooting tips below to note relevant experimental factors.
Little or no enrichment of methylated DNA
If you are planning to conduct MeDIP experiments, please visit our page and perform a search to view the published MeDIP-related products and services using our technical filters.
For research use only. Not intended for any clinical use.