5-mC | 5-methylcystosine (monclonal antibody)
AS16 3161 | clonality: monoclonal | host: mouse | reactivity:human, mouse, other species
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|Recommended dilution||1: 600 (Dot), 1-5 µg (IP), 1: 500 (IF), 1: 200-1: 1000 (FISH)|
|Expected | apparent MW|
|Predicted reactivity||mouse, broad species range|
|Not reactive in||No confirmed exceptions from predicted reactivity known at the moment|
|Additional information||To be added when available|
|Selected references||To be added when available, antibody released in February 2016.|
Dot blot: to demonstrate the specificity of the monoclonal antibody against 5-mC, a Dot blot analysis was performed using the hmC, mC and C controls: “5-hmC, 5-mC & cytosine DNA. One hundred to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the controls were spotted on a membrane (Amersham Hybond-N+). The antibody was used at a dilution of 1:600.
Immunofluorescence: human osteosarcoma (U2OS) cells were stained with the monoclonal antibody against 5-mC. Cells were fixed with 2.5% PFA in PBS for 30’, permeabilised with 0.5% Triton X-100 for 1 hour and treated with 2N HCl for 1 hour followed by 2 x 5 minutes with 0.1 M borate buffer to depurinate the DNA. After blocking with PBS containing 0.1% Triton X-100 and 1% BSA, the cells were immunofluorescently labelled with the 5-mC antibody diluted 1:500 in blocking solution, followed by a goat anti-mouse antibody conjugated to Alexa488. Panel (A): cells were immunofluorescently labelled with the 5-mC antibody (left) or with DAPI (right). Panel (B) and (C): staining of the cells with the 5-mC antibody after incubation of the antibody with 50 μM mCTP or hmCTP, respectively.
FISH: to detect methylated chromosomal regions, FISH was performed on metaphase chromosomes from HeLa cells using the monoclonal antibody against 5-mC. The cells were blocked in metaphase by treatment with colcemid (0.1 μg/ml) for 1 - 2 hours, fixed overnight at -20°C with ethanol/glacial acetic acid and treated with 2N HCl for 30’ at room temperature. Subsequently, the cells were blocked with PBS containing 1% BSA and 0.1% Triton X-100 and stained with the 5-mC antibody (left) diluted 1:1,000 in blocking solution, followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the chromosomes with DAPI. A merge of the two staining is shown to the right.
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