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Antibodies

5-Methylcytosine Antibody

Product Sizes
5-Methylcytosine Antibody
£ POA
OADC00276
Academic Pricing
Please contact us for academic pricing.
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About this Product
SKU:
OADC00276
Application:
Immunoprecipitation, Dot Blot, Immunofluorescence
Clone:
33D3
Clonality:
Monoclonal
Concentration:
1.24 ug/ml
Format:
<b>Liquid.</b> PBS containing 0.05% azide
Host:
Mouse
Isotype:
IgG1
Purification:
Protein A purified
Shipping Conditions:
Blue Ice
Storage Conditions:
Store in small aliquots at -20 to -80 C.
Avoid multiple freeze-thaw cycles.
Supplier:
Aviva Systems Biology
MeDIP results obtained with the Aviva monoclonal antibody directed against 5-mCr. MeDIP (Methylated DNA immunoprecipitation) was performed on fragmented human genomic DNA using the Aviva monoclonal antibody against 5-mC and the MagMeDIP Kit. The DNA was prepared with the GenDNA module of the MagMeDIP kit. The fragmented DNA was spiked with the internal controls present in the kit (methylated DNA (meDNA) as a positive and unmethylated DNA (unDNA) as a negative control) prior to performing the IP. QPCR was performed with optimized primer sets, included in the kit, specific for the methylated and unmethylated DNA controls, and for a known methylated (TSH2B) and unmethylated (GAPDH) genomic region. An additional internal positive and negative control locus (4994+ and 8804-, respectively) were also tested (4994+: forward primer 5'-GGGAATATAAGGAGCGCACA-3' and reverse primer 5'- TCGGTTAAAACGGTCAGGTC-3'; 8804-: forward primer 5'-CGAGGCGTGAGTTATTCCTG-3' and reverse primer 5'-CTCTTGTGGCTGAGCTCCTT-3'). Figure shows the recovery (mean of 3 experiments), expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
MeDIP-seq (methylated DNA immunoprecipitation-sequencing) with the Aviva monoclonal antibody directed against 5-mCr. Genomic DNA from E14 ES cells was sheared with the Bioruptor® to generate random fragments (size range 300 to 700 bp). One ug of the fragmented DNA was ligated to Illumina adapters and the resulting DNA was used for a standard MeDIP assay, using 2 ug of the Aviva monoclonal against 5-mC. After recovery of the methylated DNA, Illumina sequencing libraries were generated and sequenced on an Illumina Genome Analyzer according to the manufacturer's instructions. Figures 1 Genome browser views of CA simple repeat elements with read distributions specific for 5-mC at 2 gene locations (SigleC15 and Mfsd4). Visual inspection of the peak profiles in a genome browser reveals high enrichment of CA simple repeats in affinity-enriched methylated fragments after MeDIP with the Aviva 5-mC monoclonal antibody.
Immunofluorescence results obtained with the Aviva monoclonal antibody directed against 5-mCr. Figure 5A. Human HeLa cells were stained with the Aviva monoclonal antibody against 5-mC. Cells were fixed with 4% formaldehyde in PBS for 10' at room temperature, permeabilised with 0.5% Triton X-100 for 1 hour and treated with 2N HCl for 1 hour. After blocking with PBS containing 0.1% TritonX-100 and 1% BSA, the cells were immunofluorescently labeled with the 5-mC antibody (left) diluted 1:500 in blocking solution, followed by a goat anti-mouse antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. Figure 5B: Immunofluorescent staining of an interphase HeLa cell with the Aviva 5-mC antibody followed by a goat anti-mouse antibody conjugated to FITC (yellow) and with Hoechst staining (blue).
ELISA using the Aviva monoclonal antibody directed against 5-mCr. ELISA was performed using Aviva monoclonal antibody against 5-mC, diluted 1:100. The wells were coated with a serial dilution of the methylated DNA control.
Dot blot analysis using the Aviva monoclonal antibody directed against 5-mCr. To demonstrate the specificity of the Aviva antibody against 5-mC, a Dot blot analysis was performed using the hmC, mC and C controls. One hundred to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the controls were spotted on a membrane. The antibody was used at a dilution of 1:250. Figure 3 shows a high specificity of the antibody for the methylated control.
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