Anti–5–mC monoclonal antibody 33D3

MeDIP-seq (methylated DNA immunoprecipitation-sequencing) with the monoclonal antibody directed against 5-mC
Genomic DNA from E14 ES cells was sheared to generate random fragments (size range 300 to 700 bp). One μg of the fragmented DNA was ligated to Illumina adapters and the resulting DNA was used for a standard MeDIP assay, using 2 μg of the monoclonal against 5-mC (Cat. # 25207). After recovery of the methylated DNA, Illumina sequencing libraries were generated and sequenced on an Illumina Genome Analyzer according to the manufacturer’s instructions. Figure 1A and 1B show 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 5-mC monoclonal antibody.

MeDIP results obtained with the monoclonal antibody directed against 5-mC
MeDIP (Methylated DNA immunoprecipitation) was performed on fragmented human genomic DNA using the monoclonal antibody against 5-mC (Cat. # 25207). 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 2 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).

ELISA using the monoclonal antibody directed against 5-mC
ELISA was performed using monoclonal antibody against 5-mC (Cat. # 25207), diluted 1:100. The wells were coated with a serial dilution of hydroxymethylated (hmC), methylated (mC) and unmethylated (C) DNA standards.

Dot blot analysis using the monoclonal antibody directed against 5-mC
To demonstrate the specificity of the antibody against 5-mC (Cat. # 25207), a Dot blot analysis was performed using hydroxymethylated (hmC), methylated (mC) and unmethylated (C) DNA standards. 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.

Immunofluorescence results obtained with the monoclonal antibody directed against 5-mC
Figure 5A: Human HeLa cells were stained with the monoclonal antibody against 5-mC (Cat. # 25207). Cells were fixed with 4% formaldehyde in PBS for 10 min. 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 5-mC antibody followed by a goat anti-mouse antibody conjugated to FITC (yellow) and with Hoechst staining (blue).

Surface plasmon resonance (SPR) analysis of the the monoclonal antibody directed against 5-mC
A synthesized biotin-labeled 5-mC conjugate was immobilized on a CM4 BIAcore sensorchip (GE Healthcare, France). Briefly, two flowcells were prepared by sequential injections of EDC/NHS, streptavidin, and ethanolamine. One of these flowcells served as negative control (biotinylated spacer without 5-mC), while biotinylated 5-mC conjugate was injected in the other one, to obtain an immobilization level of 55 response units (RU). All SPR experiments were performed using HBS-N buffer (10 mM HEPES, 150 mM NaCl, pH 7.4) at a flow rate of 5 μl/min. Interaction assays involved injections of 3 different dilutions of the 5-mC monoclonal antibody (Cat. # 25207) over the biotinylated 5-mC conjugate and negative control surfaces, followed by a 3 min washing step with HBS-N buffer to allow dissociation of the complexes formed. At the end of each cycle, the streptavidin surface was regenerated by injection of 0.1 M citric acid, pH 3. The sensorgrams correspond to the biotinylated 5-mC conjugate surface signal subtracted with the negative control. Data from the sensorgrams that reached binding equilibrium were used for Scatchard analysis. The value of the dissociation constant (kd) obtained by global fitting and 1:1 Langmuir model is 13 ± 9 nM.