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Methylamp Methylated DNA Capture (MeDIP) Kit

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Suggested Workflow
DNA Isolation
 
 
DNA Shearing
 
 
MeDIP
 
 
PCR Analysis
 
Schematic procedure of the Methylamp™ Methylated DNA Capture Kit.
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.
Input Type: DNA
Research Area: DNA Methylation
Target Application: Immunoprecipitation
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-1015-2424 reactions $317.00 
P-1015-4848 reactions $534.00 
Availability: Usually Ships in 1 Day or Same day delivery Same Day NY Delivery 
Product Overview

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

The Methylamp™ Methylated DNA Capture (MeDIP) Kit is a complete set of optimized reagents to enrich and capture methylated DNA fragments in a convenient microplate-based format. The method, methylated DNA immunoprecipitation (meDIP), uses a monoclonal antibody specific to 5-methylcytosine to immunoprecipitate methylated genomic DNA. The enriched methylated fractions can then be used for gene-specific DNA methylation analysis on a genome wide scale. The Methylamp™ Methylated DNA Capture Kit is suitable for combining the specificity of enriched methylated DNA with qualitative and quantitative PCR, traditional sequencing and next-generation sequencing, DNA microarrays, as well as Southern blot. The kit has the following advantages and features:

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

Background Information
MeDIP is a genome-wide, large scale technique to enrich methylated DNA by isolating methylated DNA fragments with a 5-methylcytosine antibody. This technique has played an important role in helping to identify abnormally methylated genes in diseases such as cancer. With MeDIP, the immunoprecipitated fractions of methylated DNA can be determined by PCR to evaluate the methylation state of individual regions. Additionally, the purified fractions can be input to high throughput DNA detection methods such as DNA microarrays (MeDIP-chip) and next-generation sequencing (MeDIP-seq), becoming a useful approach for methylome-level analysis and for developing comprehensive profiles of DNA methylation on a genome-wide scale.

Principle & Procedure
The most common and crucial experimental factor of MeDIP that may affect its accuracy involves the quality and cross-reactivity of the 5-methylcytosine antibody used in the procedure. Epigentek overcame such a limitation through extensive research and development to create a highly efficient MeDIP kit by applying a non-cross-reactive 5mC antibody. In the kit’s assay, DNA is sheared and then added into a microplate well or multiple wells in a high throughput format. A high quality ChIP/MeDIP-grade antibody specific to methylcytosine is then used to capture methylated DNA in the wells. The antibody-captured methylated DNA is released and is finally purified through the included spin columns. The eluted methylated DNA can now be used in various down-stream applications.

Product Components

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.

Frequently Asked Q's

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 & MSDS

[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]
Product Citations

van der Wijst MG et. al. (December 2017). Experimental mitochondria-targeted DNA methylation identifies GpC methylation, not CpG methylation, as potential regulator of mitochondrial gene expression. Sci Rep. 7(1):177.

Daraei A et. al. (July 2017). Epigenetic Changes of the ESR1 Gene in Breast Tissue of Healthy Women: A Missing Link with Breast Cancer Risk Factors? Genet Test Mol Biomarkers.

Wang HD et. al. (June 2017). Detection of fetal epigenetic biomarkers through genome-wide DNA methylation study for non-invasive prenatal diagnosis. Mol Med Rep. 15(6):3989-3998.

Khakpour G et. al. (March 2017). Methylomics of breast cancer: Seeking epimarkers in peripheral blood of young subjects. Tumour Biol. 39(3):1010428317695040.

Manish Mishra; Renu A. Kowluru et. al. (October 2016). The Role of DNA Methylation in the Metabolic Memory Phenomenon Associated With the Continued Progression of Diabetic Retinopathy. IOVS. 57:5748-5757.

Xu X et. al. (March 2016). Hypoxia-induced Endothelial-Mesenchymal Transition is associated with RASAL1 promoter hypermethylation in human coronary endothelial cells. FEBS Lett.

Li W et. al. (March 2016). Epigenetics reactivation of Nrf2 in Prostate TRAMP C1 Cells by curcumin analog FN1. Chem Res Toxicol.

Tan X et. al. (January 2016). DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition. PLoS One. 11(1):e0147816.

Tan X et. al. (January 2016). High inorganic phosphate causes DNMT1 phosphorylation and subsequent fibrotic fibroblast activation. Biochem Biophys Res Commun.

Mishra M et. al. (August 2015). Epigenetic Modification of Mitochondrial DNA in the Development of Diabetic Retinopathy. Invest Ophthalmol Vis Sci. 56(9):5133-42.

Guo Y et. al. (March 2015). Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1. Biochem Pharmacol. 94(2):69-78.

Hong S et. al. (January 2015). Epigenetic regulation of genes that modulate chronic stress-induced visceral pain in the peripheral nervous system. Gastroenterology. 148(1):148-157.e7.

Tampe B et. al. (December 2014). Induction of Tet3-dependent Epigenetic Remodeling by Low-dose Hy. EBioMedicine. 1(1)

Green TJ et. al. (August 2014). Anti-viral gene induction is absent upon secondary challenge with double-stranded RNA in the Pacific oyster, Crassostrea gigas. Fish Shellfish Immunol. 39(2):492-7.

Skowronki K et. al. (July 2014). Genome-Wide Analysis in Human Colorectal Cancer Cells Reveals Ischemia-Mediated Expression of Motility Genes via DNA Hypomethylation. PLoS One. 9(7):e103243.

Wang J et. al. (July 2014). MicroRNA-152 Regulates DNA Methyltransferase 1 and Is Involved in the Development and Lactation of Mammary Glands in Dairy Cows. PLoS One. 9(7):e101358.

Michailidi C et. al. (April 2014). Genome-wide and gene-specific epigenomic platforms for hepatocellular carcinoma biomarker development trials. Gastroenterol Res Pract. 2014:597164.

Wang HD et. al. (April 2014). DNA methylation study of fetus genome through a genome-wide analysis. BMC Med Genomics. 7:18.

Koch R et. al. (April 2014). Populational equilibrium through exosome-mediated Wnt signaling in tumor progression of diffuse large B-cell lymphoma. Blood. 123(14):2189-98.

Pol Bodetto S et. al. (October 2013). Cocaine represses protein phosphatase-1Cβ through DNA methylation and Methyl-CpG Binding Protein-2 recruitment in adult rat brain. Neuropharmacology. 73:31-40.

Zhang TY et. al. (January 2013). Epigenetic mechanisms for the early environmental regulation of hippocampal glucocorticoid receptor gene expression in rodents and humans. Neuropsychopharmacology. 38(1):111-23.

Tricker PJ et. al. (June 2012). Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development. J Exp Bot. 63(10):3799-813.

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.

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.

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.

Hicks SD et. al. (September 2010). Ethanol-induced methylation of cell cycle genes in neural stem cells. J Neurochem. 114(6):1767-80.

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