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.