Please see EpiQuik Methylated DNA Immunoprecipitation Kit if you are working with nuclear extract as the starting material.


The Methylamp™ Methylated DNA Capture Kit is a complete set of essential components which enables the experimenter to use a proprietary and unique procedure/composition to enrich methylated DNA. In the assay, an antibody specific to methyl cytosine is used to immunoprecipitate methylated genomic DNA (meDIP). The enriched methylated fractions can then be used for a standard DNA detection. The entire procedure can be completed within 3 hours and produces far superior results than any competitor kits. The Methylamp™ Methylated DNA Capture Kit is suitable for combining the specificity of enriched methylated DNA with qualitative and quantitative PCR, and southern blot as well as DNA microarray.

WHY CHOOSE THE METHYLAMP™ METHYLATED DNA CAPTURE KIT?

• Highly efficient enrichment of methylated DNA: > 98%.
• The fastest procedure available, which can be finished within 3 hours.
• Strip microplate format makes the assay flexible: manual or high throughput.
• Columns for DNA purification are included: save time and reduce labor.
• Compatible with all DNA amplification-based approaches.
• Simple, reliable, and consistent assay conditions.

The Methylamp™ Methylated DNA Capture Kit contains all reagents required for carrying out a successful capture of methylated DNA from a DNA sample. Particularly, this kit includes a ChIP-grade 5-methylcytosine antibody and a negative control normal mouse IgG. DNA is sheared, added into the microwell and captured by the antibody. DNA is released from the antibody-captured methylated DNA complex, and purified through the specifically designed Fast-Spin Column. Eluted DNA can be used for various down-stream applications. SCHEMATIC PROCEDURE:

For enrichment of methylated DNA using the kit, DNA (0.5 ug) isolated from MCF-7 cells was added into the microwell. Methylated DNA was captured by 5-mC antibody prebound to the microwells. Captured methylated DNA was used for analyzing methylation level of GAPDH and MLH1 promoter with the use of primers and probes specific to GAPDH and MLH1 promoters, respectively.

MC1 (Antibody Buffer)
MC2 (Reaction Buffer)
MC3 (Wash Buffer)
MC4 (DNA Release Buffer)
MC5 (Binding Buffer)
MC6 (Elution Buffer)
Normal Mouse IgG (1 mg/ml)*
Anti-5-Methylcytosine (1mg/ml)*
Proteinase K (10 mg/ml)*
8-Well Assay Strips (with Frame)
8-Well Strip Caps
F-Spin Column
F-Collection Tube
User Guide

* For maximum recovery of the products, centrifuge the original vial after thawing prior to opening the cap.

1. What is the difference between the Methylamp Methylated DNA Capture Kit (#P-1015) and the EpiQuik Methylated DNA Immunoprecipitation Kit (Cat. No. P-2019)?
The Methylamp Methylated DNA Capture Kit is for purified DNA as the starting materials while the EpiQuik Methylated DNA Immunoprecipitation Kit is for nuclear extract as the starting materials.

2. Why are there an insufficient amount of reagents for my experiment?
This is because you may be performing additional reactions that is depleting certain reagents but leaving other ones in excess. The kit is designed for reactions with sufficient reagents. Each input DNA, positive control, and negative control would be considered 1 reaction. You should plan the number of reactions needed accordingly when using the kit.

3. How much of DNA solution can be added into MC2?
At most 1:1 (ex: 100 µl of DNA + 100 µl of MC2).

4. How much concentrated DNA should be used for IP?
500 ng - 1 µg DNA.

5. What is yield of methylated DNA if 0.5-1 µg of input DNA is added in the well?
2-4% of total input that is, 10-20 ng.

6. How can I enrich 200 ng of mDNA, as required for a microarray?
Pool the IPed mDNA from 10-20 wells and then purify the mDNA with 5-10 columns (2 wells/each column). It is also useful to generate sufficient DNA for a microarray by amplify IPed mDNA using whole genome amplification (WGA).

[User Guide]* *Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

[Material Safety Data Sheet]

Yu, F. et. al. (January 2012). MicroRNA 34c Gene Down-regulation via DNA Methylation Promotes Self-renewal and Epithelial-Mesenchymal Transition in Breast Tumor-initiating Cells. J.Biol Chem. 287 (1):465-73. PubMed Abstract

Korostowski, L. et. al. (November 2011). Enhancer-driven chromatin interactions during development promote escape from silencing by a long non-coding RNA. Epigenetics Chromatin. 4:21. PubMed Abstract

Zhou, FC. et. al. (April 2011). Alcohol alters DNA methylation patterns and inhibits neural stem cell differentiation. Alcohol Clin Exp Res. 35(4):735-46. PubMed Abstract

Hicks, S. D. et. al. (September 2010). Ethanol-induced methylation of cell cycle genes in neural stem cells. Journal of Neurochemistry. 114(6): 1767-80. PubMed Abstract