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![Immunocytochemistry/Immunofluorescence: 5-MethylCytosine Antibody (33D3) [NBP2-54609] - HeLa cells were stained with the antibody against 5-mC and with DAPI. Cells were fixed with 4% formaldehyde for 10 minutes and blocked with PBS/TX-100 containing 1% BSA. The cells were immunofluorescently labelled with the 5-mC antibody (middle) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa Fluor 594. The left panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.](http://images.novusbio.com/fullsize/5-MethylCytosine-Antibody-33D3-Immunocytochemistry-Immunofluorescence-NBP2-54609-img0022.jpg "Immunocytochemistry/Immunofluorescence: 5-MethylCytosine Antibody (33D3) [NBP2-54609] - HeLa cells were stained with the antibody against 5-mC and with DAPI. Cells were fixed with 4% formaldehyde for 10 minutes and blocked with PBS/TX-100 containing 1% BSA. The cells were immunofluorescently labelled with the 5-mC antibody (middle) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa Fluor 594. The left panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.")
![Surface Plasmon Resonance: 5-MethylCytosine Antibody (33D3) [NBP2-54609] - A synthesized biotin-labeled 5-mC conjugate was immobilized on a sensorchip. Briefly, two flowcells were prepared by sequential injections of EDC/NHS, streptavidin, and ethanolamine. One of these flowcells served as negative control, while biotinylated 5-mC conjugate was injected in the other one, to get 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 uL/min. Interaction assays involved injections of 2 different dilutions of the 5-mC monoclonal antibody over the biotinylated 5-mC conjugate and negative control surfaces, followed by a 3 minute washing step with HBS-N buffer. At the end of each cycle, the streptavidin surface was regenerated by injection of 0.1M citric acid (pH 3). The value of the dissociation constant (kd) obtained by global fitting and 1:1 Langmuir model is 65 nM.](http://images.novusbio.com/fullsize/5-MethylCytosine-Antibody-33D3-Ovalbumine-Surface-Plasmon-Resonance-NBP2-54609-img0015.jpg)
![Dot Blot: 5-MethylCytosine Antibody (33D3) [NBP2-54609] - To demonstrate the specificity of the antibody against 5-mC, a Dot blot analysis was performed using hmC, mC and C controls. 100 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 1:300. Figure shows a high specificity of the antibody for the methylated control.](http://images.novusbio.com/fullsize/5-MethylCytosine-Antibody-33D3-Dot-Blot-NBP2-54609-img0018.jpg)
![Methylated DNA Immunoprecipitation: 5-MethylCytosine Antibody (33D3) [NBP2-54609] - Analysis was performed on 1 ug fragmented human genomic DNA using 0.2 ug of the monoclonal antibody against 5-mC. The fragmented DNA was spiked with controls (methylated DNA (meDNA) as a positive and unmethylated DNA (unDNA) as a negative control) prior to performing the IP. QPCR was performed with primer sets specific for the methylated and unmethylated DNA controls, and for a known methylated (TSH2B) and unmethylated (GAPDH) genomic region. The figure shows the recovery expressed as a percent of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).](http://images.novusbio.com/fullsize/5-MethylCytosine-Antibody-33D3-Methylated-DNA-Immunoprecipitation-NBP2-54609-img0023.jpg)