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5-Methylcytosine (5-mC) Monoclonal Antibody [33D3]


For antibody-based applications requiring high specificity to methylated cytosines

Citations (77) | (2) | Write a Review
Immunofluorescence staining with 5-methylcytosine antibody (anti-5mc) clone 33D3.
Methylated DNA successfully captured by 5-methylcytosine antibody (anti-5mc) clone 33D3 during MeDIP. Fig. 2. Immunofluorescence staining with 5-methylcytosine antibody (anti-5mc) clone 33D3.
Synthesized oligos containing different numbers of 5-methylcytosines were captured with the Clone 33D3 anti-5-methylcytosine antibody (Cat No. A-1014) and then colorimetrically detected. The results show that the oligos containing as few as two 5-mCs can still be captured and oligos with four or more 5-mCs can be fully captured by the antibody.
Synthesized oligos containing different numbers of 5-methylcytosines were captured with anti-5-methylcytosine antibodies from various companies and then colorimetrically detected for comparison. Results show Epigentek's 5-mC antibody has the highest sensitivity and specificity in capturing methylated DNA fragments.
Application: DB, ELISA, IF, IHC, IP, MeDIP
Areas of Research: DNA Methylation
Clonality: Monoclonal
Conjugate: Unconjugated
Host: Mouse
Isotype: IgG1
Purification: Affinity Purified
Reactivity: Broad Range
Trial Size Available: Yes
100% Guarantee: 6 months
Catalog No.SizePriceQty
A-1014-01010 µg $69.00 
A-1014-05050 µg $199.00 
A-1014-100100 µg $380.00 
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Product Overview

5-methylcytosine (5-mC) is formed when DNA methyltransferase (DNMT) catalyzes the addition of a methyl group onto the 5-carbon of the cytosine ring, an epigenetic process known as DNA methylation. 5-mC is considered the "fifth" DNA base and this 5-methylcytosine mouse monoclonal antibody is ideal for discriminating between the unmodified cytosine base (C) and the methylated cytosine base (5-mC) for DNA methylation studies. DNA methylation, the major epigenetic modification of eukaryotic genomes, plays an essential role in mammalian development. DNA methylation of promoter regions leads to inactivation of gene function. Also, DNA methylation status varies according to tissue type, and region-specific DNA hypermethylation and global DNA hypomethylation have been demonstrated to play an important role in tumorigenesis. 

Mouse monoclonal antibody to 5-methylcytosine (5-mC), clone 33D3, MeDIP/ChIP-grade, used in DNA methylation studies.

1 mg/ml

Protein A

Ovalbumin-conjugated 5-methylcytosine (5-mC)


Modified base 5-methylcytosine (5-mC), a broad range of species.

Purified IgG in 10 mM phosphate buffer, 150 mM NaCl, pH 7.4.

4°C, stable for 6 months from the date of shipment. For long-term storage, aliquot and store at -20°C. Avoid repeated freezing and thawing. Multiple freeze/thaw cycles may result in decreased performance.

Handling Recommendations
For maximum recovery of the products, centrifuge the vial prior to opening the cap.

Alternative Names
5-methylcytidine, anti-5-methylcytidine, anti-5-methylcytosine, anti-5mC, anti-5-mC, anti-5meC, anti-5-meC, 5mC, 5meC, 5-meC, 5'-methyl-2'-deoxycytidine, 5MedCyd

Application & Suggested Dilutions*
Dot Blot: 1:1000-1:2000; Immunohistochemistry: 1:100-1:500; Immunofluorescence: 1:100-1:500; ELISA: 1:1000-1:2000; MeDIP: 0.5-1 µg/reaction

*The end user is responsible for determining optimal working dilutions.

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.

Product Citations

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Kurinomaru T et. al. (September 2019). Sequential Assessment of Multiple Epigenetic Modifications of Cytosine in Whole Genomic DNA by Surface Plasmon Resonance. Anal Chem.

Poplawski S.G et. al. (August 2019). An antisense oligonucleotide leads to suppressed transcriptional elongation of Hdac2 and long-term memory enhancement bioRxiv.

Catania S et. al. (July 2019). Evolutionary persistence of DNA methylation for millions of years after ancient loss of a de novo methyltransferase bioRxiv.

Yamada T et. al. (April 2019). UV irradiation-induced DNA hypomethylation around WNT1 gene: implications for solar lentigines. Exp Dermatol.

Greer C et. al. et. al. (April 2019). An antisense oligonucleotide leads to suppressed transcriptional elongation of Hdac2 and long-term memory enhancement biorxiv.

Kalle A.M. et. al. (March 2019). Differential effects of two HDAC inhibitors with distinct concomitant DNA hypermethylation or hypomethylation in breast cancer cells bioRxiv.

Wei Xu et. al. (February 2019). Genome-Wide Plasma Cell-Free DNA Methylation Profiling Identifies Potential Biomarkers for Lung Cancer Disease Markers. 2019

Vincenzetti L et. al. (January 2019). The contribution of active and passive mechanisms of 5mC and 5hmC removal in human T lymphocytes is differentiation- and activation-dependent. Eur J Immunol.

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Schulze M et. al. (July 2018). Sporadic Parkinson's disease derived neuronal cells show disease-specific mRNA and small RNA signatures with abundant deregulation of piRNAs. Acta Neuropathol Commun. 6(1):58.

Ceccarelli V et. al. (May 2018). Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism. FASEB J. :fj201800245R.

Zhou D et. al. (March 2018). Distinctive epigenomes characterize glioma stem cells and their response to differentiation cues. Genome Biol. 19(1):43.

Fang Y et. al. (February 2018). Melatonin-Mediated Development of Ovine Cumulus Cells, Perhaps by Regulation of DNA Methylation. Molecules. 23(2)

Luo N et. al. (January 2018). DNA methyltransferase inhibition upregulates MHC-I to potentiate cytotoxic T lymphocyte responses in breast cancer. Nat Commun. 9(1):248.

You D et. al. (November 2017). Dnmt3a is an epigenetic mediator of adipose insulin resistance. Elife. 6

Rajabi H et. al. (August 2017). The influence of semen quality on male pronucleus demethylation process during ICSI cycle. Syst Biol Reprod Med. :1-9.

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Kumar D et. al. (May 2017). Effect of perinatal exposure to Bisphenol-A on DNA methylation and histone acetylation in cerebral cortex and hippocampus of postnatal male mice. J Toxicol Sci. 42(3):281-289.

Rwigemera A et. al. (April 2017). Fetal testis organ culture reproduces the dynamics of epigenetic reprogramming in rat gonocytes BioMed Central. 10(19)

Maag JL et. al. (March 2017). Widespread promoter methylation of synaptic plasticity genes in long-term potentiation in the adult brain in vivo. BMC Genomics. 18(1):250.

Qu K et. al. (February 2017). Vitamin C down-regulate apo(a) expression via Tet2-dependent DNA demethylation in HepG2 cells. Int J Biol Macromol.

Leoni C et. al. (February 2017). Dnmt3a restrains mast cell inflammatory responses. Proc Natl Acad Sci U S A.

David R et. al. (January 2017). Transcriptome-wide Mapping of RNA 5-Methylcytosine in Arabidopsis mRNAs and non-coding RNAs. Plant Cell.

Liu MH et. al. (August 2016). Ultrastructural Changes and Methylation of Human Oocytes Vitrified at the Germinal Vesicle Stage and Matured in vitro after Thawing. Gynecol Obstet Invest.

Savell KE et. al. (July 2016). Extra-coding RNAs regulate neuronal DNA methylation dynamics. Nat Commun. 7:12091.

Timoshevskiy VA et. al. (June 2016). Cellular and Molecular Features of Developmentally Programmed Genome Rearrangement in a Vertebrate (Sea Lamprey: Petromyzon marinus). PLoS Genet. 12(6):e1006103.

Rajabi H et. al. (February 2016). Pronuclear epigenetic modification of protamine deficient human sperm following injection into mouse oocytes. Syst Biol Reprod Med. :1-8.

Heyward FD et. al. (January 2016). Obesity Weighs down Memory through a Mechanism Involving the Neuroepigenetic Dysregulation of Sirt1. J Neurosci. 36(4):1324-35.

Costa G et. al. (January 2016). DNA demethylation caused by 5-Aza-2'-deoxycytidine induces mitotic alterations and aneuploidy. Oncotarget. 7(4):3726-39.

Karpova NN et. al. (January 2016). Protocol for Methylated DNA Immunoprecipitation (MeDIP) Analysis Epigenetic Methods in Neuroscience Research. 105:97-114.

Snyder MD et. al. (December 2015). Suppression of ASH2L alters DNA methylation and histone patterns during bovine embryonic development. Reprod Fertil Dev. 28(2):131.

Golding MC et. al. (July 2015). Histone-lysine N-methyltransferase SETDB1 is required for development of the bovine blastocyst. Theriogenology.

Wu W et. al. (July 2015). A Cell Electrofusion Chip for Somatic Cells Reprogramming. PLoS One. 10(7):e0131966.

Kim SH et. al. (June 2015). Egg-specific expression of protein with DNA methyltransferase activity in the biocarcinogenic liver fluke Clonorchis sinensis. Parasitology. :1-11.

Singh P et. al. (May 2015). Hippocampal chromatin modifying enzymes are pivotal for scopolamine- induced synaptic plasticity gene expression changes and memory impairment. J Neurochem.

Zembrzycki A et. al. (May 2015). Postmitotic regulation of sensory area patterning in the mammalian neocortex by Lhx2. Proc Natl Acad Sci U S A.

Winans B et. al. (May 2015). Linking the Aryl Hydrocarbon Receptor with Altered DNA Methylation Patterns and Developmentally Induced Aberrant Antiviral CD8+ T Cell Responses. J Immunol. 194(9):4446-57.

Nguyen AH et. al. (April 2015). Gold nanostar based biosensor detects epigenetic alterations on promoter of real cells. Biosens Bioelectron. 66:497-503.

Fang Y et. al. (March 2015). Effects of the TLR4 transgene on reproductive traits and DNA methylation pattern of oocytes in ewes Front Agr Sci Eng. 1(4):314-20.

Leoni C et. al. (February 2015). Reduced DNA methylation and hydroxymethylation in patients with systemic mastocytosis. Eur J Haematol.

Jia G et. al. (December 2014). Spermatozoa cryopreservation alters pronuclear formation and zygotic DNA demethylation in mice. Theriogenology.

Zovkic IB et. al. (September 2014). Histone H2A.Z subunit exchange controls consolidation of recent and remote memory. Nature.

Nguyen AH et. al. (September 2014). Nanoplasmonic biosensor: Detection and amplification of dual bio-signatures of circulating tumor DNA. Biosens Bioelectron.

Heras S et. al. (June 2014). DNA counterstaining for methylation and hydroxymethylation immunostaining in bovine zygotes. Anal Biochem. 454:14-6.

Wang Y et. al. (April 2014). Hypermethylation of the enolase gene (ENO2) in autism. Eur J Pediatr.

Pierron F et. al. (January 2014). Effect of low-dose cadmium exposure on DNA methylation in the endangered European eel. Environ Sci Technol. 48(1):797-803.

Yu Z et. al. (October 2013). Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting duct. Am J Physiol Renal Physiol. 305(7):F1006-13.

Day JJ et. al. (October 2013). DNA methylation regulates associative reward learning. Nat Neurosci. 16(10):1445-52.

Carbone DL et. al. (June 2013). Sex and stress hormone influences on the expression and activity of brain-derived neurotrophic factor. Neuroscience. 239:295-303.

Zhang L et. al. (February 2013). Tet-mediated covalent labelling of 5-methylcytosine for its genome-wide detection and sequencing. Nat Commun. 4:1517.

Sato A et. al. (February 2013). CCAAT/enhancer-binding protein-α suppresses lung tumor development in mice through the p38α MAP kinase pathway. PLoS One. 8(2):e57013.

Zhang X et. al. (January 2013). Promoter hypermethylation of ARID1A gene is responsible for its low mRNA expression in many invasive breast cancers. PLoS One. 8(1):e53931.

Liang Y et. al. (October 2012). DNA methylation pattern in mouse oocytes and their in vitro fertilized early embryos: effect of oocyte vitrification. Zygote. :1-8.

Huang SK et. al. (September 2012). Prostaglandin E₂ increases fibroblast gene-specific and global DNA methylation via increased DNA methyltransferase expression. FASEB J. 26(9):3703-14.

Chen L et. al. (July 2012). Naturally occurring endo-siRNA silences LINE-1 retrotransposons in human cells through DNA methylation. Epigenetics. 7(7):758-71.

Yue MX et. al. (July 2012). Abnormal DNA methylation in oocytes could be associated with a decrease in reproductive potential in old mice. J Assist Reprod Genet. 29(7):643-50.

Yu M et. al. (June 2012). Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell. 149(6):1368-80.

Li Y et. al. (May 2012). Plasticity of DNA methylation in mouse T cell activation and differentiation. BMC Mol Biol. 13:16.

Ge C et. al. (May 2012). A simple colorimetric detection of DNA methylation. Analyst. 137(9):2032-5.

Li Y et. al. (March 2012). IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages. Biochem Biophys Res Commun. 419(4):748-53.

Barra V et. al. (February 2012). Bypass of cell cycle arrest induced by transient DNMT1 post-transcriptional silencing triggers aneuploidy in human cells. Cell Div. 7(1):2.

Zhang Y et. al. (October 2011). Effects of DNMT1 silencing on malignant phenotype and methylated gene expression in cervical cancer cells. J Exp Clin Cancer Res. 30:98.

Aoi Y et. al. (October 2011). The involvement of DNA and histone methylation in the repression of IL-1β-induced MCP-1 production by hypoxia. Biochem Biophys Res Commun. 414(1):252-8.

Li L et. al. (July 2011). Genome-wide DNA methylation patterns in IVF-conceived mice and their progeny: a putative model for ART-conceived humans. Reprod Toxicol. 32(1):98-105.

Liu S et. al. (March 2011). Effect of gonadotropins on dynamic events and global deoxyribonucleic acid methylation during in vitro maturation of oocytes: an animal model. Fertil Steril. 95(4):1503-6.e1-3.

Mandrioli M et. al. (January 2011). Composition and epigenetic markers of heterochromatin in the aphid Aphis nerii (Hemiptera: Aphididae). Cytogenet Genome Res. 133(1):67-77.

Yang Y et. al. (October 2010). Identification of methylated regions with peak search based on Poisson model from massively parallel methylated DNA immunoprecipitation-sequencing data. Electrophoresis. 31(21):3537-44.

Kim MS et. al. (October 2009). DNA demethylation in hormone-induced transcriptional derepression. Nature. 461(7266):1007-12.

Weng YI et. al. (September 2009). Methylated DNA immunoprecipitation and microarray-based analysis: detection of DNA methylation in breast cancer cell lines. Methods Mol Biol. 590:165-76.

Chen C et. al. (June 2009). Aberrant DNA methylation in thymic epithelial tumors. Cancer Invest. 27(5):582-91.

Tsuji Y et. al. (May 2009). The developmental potential of mouse somatic cell nuclear-transferred oocytes treated with trichostatin A and 5-aza-2'-deoxycytidine. Zygote. 17(2):109-15.

Roth TL et. al. (May 2009). Lasting epigenetic influence of early-life adversity on the BDNF gene. Biol Psychiatry. 65(9):760-9.

Yun XX et. al. (April 2009). DNA methylation patterns of mouse tetraploid embryos. Yi Chuan. 31(4):387-92.

Lubin FD et. al. (October 2008). Epigenetic regulation of BDNF gene transcription in the consolidation of fear memory. J Neurosci. 28(42):10576-86.

Novikova SI et. al. (April 2008). Maternal cocaine administration in mice alters DNA methylation and gene expression in hippocampal neurons of neonatal and prepubertal offspring. PLoS One. 3(4):e1919.

Mandrioli M et. al. (November 2007). Analysis of heterochromatic epigenetic markers in the holocentric chromosomes of the aphid Acyrthosiphon pisum. Chromosome Res. 15(8):1015-22.

Customer Reviews

Rating by s*****@uab.edu Verified Purchase Reviewed on: Thursday 22 December, 2016
Application Description
Pros: Only small amount of antibody needed per sample.

Cons: Must denature DNA before using- not mentioned in user manual that it binds single stranded DNA.

Procedural Details
In vitro methylation assay: In vitro methylation assays were performed with 10 ng of a 189-bp gDNA template from the rat Fos promoter (containing 15 CpG sites). A control 5mC strand was created by mixing in methylated dNTPs into PCR reaction. DNA was incubated for 1 h at 37 °C with 0.06mM DNMT3a-3l (Sigma, SRP0396) and 0.5mM S-(50Adenosyl)-L- methyonine (SAM, Sigma) in methylation buffer alongside DNA, protein and SAM only controls. MeDIP was carried out with 10 ul magnetic beads (Protein-A coated Dynabeads Beads, Invitrogen) and 1 ul 5 mC antibody (Epigentek, #A-1014) for 1 h at 4 °C with rotation on ssDNA. Beads were washed 3x with 1 bind/wash buffer for 5 min/wash and re-suspended in buffer EB with 1% SDS. Proteinase K (Qiagen) was added and reactions were incubated at 50 °C for 1 h then 95 °C for 10 min to reverse binding. Beads were removed and IP reactions and inputs were purified using a PCR Purification Kit (Qiagen). Purified DNA was subjected to qPCR, and methylation levels were computed as a fraction of input DNA.

Other Thoughts
Please include in user guide that DNA must be single stranded to bind for applications such as MeDIP.

Co-incubation of DNMT3a/3l protein and the methyl donor SAM results in cytosine methylation at dsDNA from the rat Fos promoter.
Rating by n*****@vanderbilt.edu Verified Purchase Reviewed on: Thursday 22 December, 2016
Application Description
The product is used for detecting DNA methylation level. The DNA was denatured and cross-linked to the membrane. Then incubate with methylcytosine antibody for 1h at RT. After incubation with secondary antibody, developed signal with ECL.

Pros: the steps are simple & the antibody is sensitive.
Cons: not for this one.

Procedural Details
Here is the picture shows that the antibody has a superior sensitivity to 5-methylcytosine over 5-hydroxymethlcytosine. NO detection to cytosine. Also, the antibody can even detect 1ng/ul 5-methylcytosine.

Other Thoughts
the antibody works pretty good to detect DNA methylation

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