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

Catalog#: AMM99021

• Size: 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.
• Host: Mouse
• Application: WB, ELISA, FC, IHC-FP, RIA
• Clone: 33D3
• Product Format: 1 mg/ml in 10mM phosphate buffer; 150 mM NaCl; pH 7.4
• Isotype: Mouse IgG1
• Reconstitution and Storage: Ready for usage. For longer periods of storage, store at -20°C. Avoid repeat freeze-thaw cycles.
• Key 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

Citation:
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.