Anti-5-methyl cytosine AMM99021 has recently been referenced in the following publications:
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
Dhawan, S., Georgia, S., Tschen, S.-I., Fan, G. & Bhushan, A. Pancreatic β cell identity is maintained by DNA methylation-mediated repression of Arx. Dev. Cell 20, 419–29 (2011). IF/ICC, Mouse 21497756
- 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.
- WB, ELISA, FC, IHC-FP, RIA
- 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.
- Key Reference:
- Maki W. C., et al., Biosens Bioelectron 23(6):780-787 (2008)
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
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.
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)
Researcher: Chinweike Ukomadu, Brigham and Women's Hospital, Boston
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)
Washed 3X, 5 min each in PBST
Washed 3X, 5 min each in PBST
Developed by ECL
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Aviva Systems Biology