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5-Methylcytosine (Methyl-CpG) Antibody (Clone #: 33D3)(50ug)

Description of Target:
Methylation of DNA cytosine bases plays important roles in the regulation of gene transcription. It has been estimated that 60 to 90% of the cytosines in CpG dinucleotides are methylated. An increase in methyl-CpG correlates with transcriptional silencing for the whole chromosome especially in developmentally regulated genes. Methylation is believed to lead to transcriptional silencing by multiple mechanisms including alteration of transcription factor binding to promoters and alteration in chromatin structure.
Product Format:
1 mg/ml in 10mM phosphate buffer; 150 mM NaCl; pH 7.4
Mouse IgG1
Datasheets / Downloads:
Species Reactivity:
Drosophila Melanogaster; Human; Arabidopsis; Zebra fish; Dog; Mouse; C.elegans; Rat; Pig; Chicken
Reconstitution and Storage:
Ready for usage. For longer periods of storage, store at -20°C. Avoid repeat freeze-thaw cycles.

Anti-5-methyl cytosine AMM99021 has recently been referenced in the following publications:

Zhang, W., Lee, H.-R., Koo, D.-H. & Jiang, J. Epigenetic modification of centromeric chromatin: hypomethylation of DNA sequences in the CENH3-associated chromatin in Arabidopsis thaliana and maize. Plant Cell 20, 25–34 (2008). ICC/IF, Arabidopsis 18239133

Maki, W. C. et al. Nanowire-transistor based ultra-sensitive DNA methylation detection. Biosens. Bioelectron. 23, 780–7 (2008). ChIP, Human 17936611

Gertych, A., Oh, J. H., Wawrowsky, K. A., Weisenberger, D. J. & Tajbakhsh, J. 3-D DNA methylation phenotypes correlate with cytotoxicity levels in prostate and liver cancer cell models. BMC Pharmacol. Toxicol. 14, 11 (2013). ICC/IF, Human 23394161

Minardi, D. et al. Prognostic role of global DNA-methylation and histone acetylation in pT1a clear cell renal carcinoma in partial nephrectomy specimens. J. Cell. Mol. Med. 13, 2115–21 (2009). IHC, Human 18752633

Jensen, T. J., Novak, P., Eblin, K. E., Gandolfi, A. J. & Futscher, B. W. Epigenetic remodeling during arsenical-induced malignant transformation. Carcinogenesis 29, 1500–8 (2008). IP, Human 18448484

Vrba, L., Junk, D. J., Novak, P. & Futscher, B. W. p53 induces distinct epigenetic states at its direct target promoters. BMC Genomics 9, 486 (2008). IP, Human 18922183

Macaulay, E. C., Weeks, R. J., Andrews, S. & Morison, I. M. Hypomethylation of functional retrotransposon-derived genes in the human placenta. Mamm. Genome 22, 722–35 (2011). IP, Human 21874386

Fukuda, K. et al. Regional DNA methylation differences between humans and chimpanzees are associated with genetic changes, transcriptional divergence and disease genes. J. Hum. Genet. 58, 446–54 (2013). IP, Human, Chimpanzee 23739127

Ribas, R. et al. Members of the TEAD family of transcription factors regulate the expression of Myf5 in ventral somitic compartments. Dev. Biol. 355, 372–80 (2011). ICC/IF, Mouse 21497756

Zhang, W., Wang, X., Yu, Q., Ming, R. & Jiang, J. DNA methylation and heterochromatinization in the male-specific region of the primitive Y chromosome of papaya. Genome Res. 18, 1938–43 (2008). FISH, Papaya 18593814

Walsh, T. K. et al. A functional DNA methylation system in the pea aphid, Acyrthosiphon pisum. Insect Mol. Biol. 19 Suppl 2, 215–28 (2010). IP, Pea aphid 20482652

Kurita, R., Arai, K., Nakamoto, K., Kato, D. & Niwa, O. Determination of DNA methylation using electrochemiluminescence with surface accumulable coreactant. Anal. Chem. 84, 1799–803 (2012). 22263690

Kurita, R. & Niwa, O. DNA methylation analysis triggered by bulge specific immuno-recognition. Anal. Chem. 84, 7533–8 (2012). 22880797

Tajbakhsh, J. Covisualization of methylcytosine, global DNA, and protein biomarkers for In Situ 3D DNA methylation phenotyping of stem cells. Methods Mol. Biol. 1052, 77–88 (2013). 23592032

Target Reference:
Maki W. C., et al., Biosens Bioelectron 23(6):780-787 (2008)

Product Protocols: Immunodetection of 5-methylcytosine Protocol (Catalog Number: AMM99021)

Product Protocols:
Immunodetection of 5-methylcytosine Protocol with Antibody (Catalog Number: AMM99021)

Catalog Number: AMM99021

1. Zhang W, Wang X, Yu Q, Ming R, Jiang J. DNA methylation and heterochromatinization in the male-specific region of the primitive Y chromosome of papaya. Genome Res. 2008 Dec,18(12):1938-43. Epub 2008 Jul 1. PubMed PMID: 18593814, PubMed Central PMCID: PMC2593574.

Product Name: 5-Methylcytosine (Methyl-CpG) Antibody (Clone #: 33D3)(50ug)
Species: Two Hawaiian gynodioecious papaya cultivars, Kapoho and SunUp

Experiment Name: Immunodetection of 5-methylcytosine

Experiment Background:
1. Here, Wenli et al. reported that the male-specific region of the Y chromosome (MSY) spans ∼13% of the papaya Y chromosome. Interestingly, the centromere of the Y chromosome is embedded in the MSY. They observed four knob-like heterochromatin structures specific to the MSY.
2. They conducted immunofluorescence assays using an antibody against 5-methylcytosine (5mC) to investigate whether the knob-related DNA sequences in the MSY are more heavily methylated than the sequences located in the corresponding X chromosomal regions.

Experimental Steps:
1. Chromosome preparations were the same as FISH procedure.
2. After denaturing in 70% formamide containing 2× SSC for 3 min at 80 C, the slides were washed in ice-cold 70% ethanol for 5 min and incubated in the blocking reaction (1× PBS containing 1% bovine serum albumin and 0.5% Tween 20) for 30 min at 37 C in a wet chamber.
3. The mouse antiserum raised against 5-methylcytosine (Aviva Systems Biology) was diluted by 1:250 in 1× TNB (100 mM Tris HCl at pH 7.5, 150 mM NaCl, 0.5% blocking reagent) and applied to the slides and kept in a humid chamber for 5 h at 37 C.
4. After washing in 1× PBS three times for 5 min, FITC-labeled goat anti-mouse IgG (Jackson Immunoresearch Lab) was applied as the secondary antibody.
5. Chromosomes were counterstained with DAPI. After recording the 5mC signals, the slides were dipped in 1× PBS buffer to remove the coverglasses, and dehydrated in an ethanol series.
6. The MSY region was identified by probing with an MSY-specific BAC clone 99O03.
7. Quantification of fluorescence from DAPI staining and 5mC immunofluorescence was performed following published protocols.

Product Review: 5-Methylcytosine (Methyl-CpG) Antibody (Clone #: 33D3) in gRNA from HepG2 using DB (AMM99021)

Product Page for 5-Methylcytosine (Methyl-CpG) Antibody (Clone #: 33D3)(50ug)

Researcher: Chinweike Ukomadu, Brigham and Women's Hospital, Boston
Application: DB
Species+tissue/cell type: gRNA from HepG2
Primary antibody dilution:1:1000
Secondary antibody: Anti rabbit-HRP
Secondary antibody dilution: 1:5,000

How do Aviva’s reagents play a role in your experimental goals? We need a good reagent against 5-MeC
How would you rate this antibody on a scale from 1-5 (5=best) nad why? 5
Would you use this antibody in future experiment? Absolutely
Have you used another antibody which has worked in your application? Yes
Do you believe the information about the reagent on Aviva’s website is correct? Yes
If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not? Yes. It is a great antibody.
How did you store the antibody after re-suspension? 4 degrees C
Sample Description (please include 1) species type, and 2) cell/tissue type, and 3) how much protein loaded for each lane of your gel): HepG2 cells
How many different experimental trials were conducted using the antibody sample? >2
How was this sample prepared? Genomic DNA was prepared from cells. 100 ng od DNA per sample was denatured in 0.4 M NaOH, 10 mM EDTA at 95 degrees for 10 min and then neutralized in an equal volume of cold 2M ammonium acetate. Samples were applied to slot blotter. Sample were then processed as per Western blots
Primary antibody dilution and incubation time: 1:1000, overnight 4 degrees
Secondary antibody used and dilution and incubation time: 1: 5000, 1 hour
What controls were used in your experiment (positive/negative)? Positive and negative controls as noted in attached picture
Please include your detailed WB Procedure/Protocol here: Block for 1 hour 5% non fat milk in 0.1 Tween-20 in PBS (PBST)
Added primary
Washed 3X, 5 min each in PBST
Added secondary
Washed 3X, 5 min each in PBST
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