5-mC | 5-methylcystosine (monclonal antibody for MeDIP/IF)
AS16 3162 | Clonality: monoclonal | Host: Mouse | Reactivity:Human, Mouse, other species
|Recommended dilution||05-1 µg (MeDIP), 1: 100 (IF)|
|Expected | apparent MW|
|Predicted reactivity||Mouse, plants, broad species range|
|Not reactive in||No confirmed exceptions from predicted reactivity are currently known.|
|Selected references||To be added when available, antibody released in February 2016.|
MeDIP was performed using 1 μg fragmented genomic DNA isolated from human blood, the monoclonal antibody against 5-mC and optimized PCR primer sets for qPCR of the indicated regions. 0.2 μg of antibody was used per IP experiment. The graph shows the recovery (expressed as a % of the input DNA, mean of 4 experiments) of the TSH2B gene, known to be methylated and of the promoter of the active GAPDH gene, used as a negative control.
Chart flow showing steps of Methylated DNA-immunoprecipiation (MeDIP) method:
- Prepare genomic DNA from cultured cells
- Shear genomic DNA
- Denature the sheared genomic DNA
- Immunoprecipiate with the antibody against 5-meC
- Isolate DNA and perform PCR
Immunofluorescence: HeLa cells were stained with the antibody against 5-mC and with DAPI. Cells were fixed with 2.5% formaldehyde for 30’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the 5-mC antibody (left) diluted 1:100 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.
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