5-MethylCytosine Antibody (33D3)

Immunocytochemistry/ Immunofluorescence: 5-MethylCytosine Antibody (33D3) [NBP2-54609]

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]

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.

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.