![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.")
| Reactivity | Hu, Mu, Rt, BvSpecies Glossary |
| Applications | DB, ICC/IF, IP, ICC/IF, SPR |
| Clone | 33D3 |
| Clonality | Monoclonal |
| Host | Mouse |
| Conjugate | Unconjugated |
| Format | BSA Free |
| Description | Novus Biologicals Mouse 5-MethylCytosine Antibody (33D3) - BSA Free (NBP2-54609) is a monoclonal antibody validated for use in ICC/IF. All Novus Biologicals antibodies are covered by our 100% guarantee. |
| Immunogen | 5-methylcytosine |
| Isotype | IgG1 |
| Clonality | Monoclonal |
| Host | Mouse |
| Purity | Protein A purified |
| Innovator's Reward | Test in a species/application not listed above to receive a full credit towards a future purchase. |
| Dilutions |
|
| Storage | Store at -80C. Avoid freeze-thaw cycles. |
| Buffer | PBS |
| Preservative | 0.05% Sodium Azide |
| Purity | Protein A purified |
Secondary Antibodies |
Isotype Controls |
The concentration calculator allows you to quickly calculate the volume, mass or concentration of your vial. Simply enter your mass, volume, or concentration values for your reagent and the calculator will determine the rest.
![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 "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.](http://images.novusbio.com/fullsize/5-MethylCytosine-Antibody-33D3-Dot-Blot-NBP2-54609-img0018.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.")
![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 "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).")
![SDS-Page: Mouse IgG1 Isotype Control (MG1) [NBP1-97005] - Lane 1: , Non-reduced. M: Opal Pre-stained Ladder. Lane 2: , Reduced. Load: 1.0 ug per lane. Predicted/Observed: 120 kDa Non-reduced, 55 and 25 Reduced.](https://images.novusbio.com/images/Mouse-IgG1-Isotype-Control-MG1-SDS-Page-NBP1-97005-img0002.jpg "SDS-Page: Mouse IgG1 Isotype Control (MG1) [NBP1-97005] - Lane 1: , Non-reduced. M: Opal Pre-stained Ladder. Lane 2: , Reduced. Load: 1.0 ug per lane. Predicted/Observed: 120 kDa Non-reduced, 55 and 25 Reduced.")