Anti-5-Methylcytosine Species ReactivityReacts with methylated DNA from a broad range of species. 1 unit

Data sheet

5-Methylcytosine Antibody

AMM99021

Description: 

Methylation of DNA cytosine bases plays important roles in the regulation of gene transcription. It has been estimated that 60 to 90% of the cytosines in CpG dinucleotides are methylated. An increase in methyl-CpG correlates with transcriptional silencing for the whole chromosome especially in developmentally regulated genes. Methylation is believed to lead to transcriptional silencing by multiple mechanisms including alteration of transcription factor binding to promoters and alteration in chromatin structure. Clone number 33D3 is useful in several applications including: ELISA (0.1 µg/ml), Flow Cytometry (1 µg/ml), Cryo Sections (5-10 µg/ml), Paraffin Sections (5-10 µg/ml), and RIA (0.1 µg/ml).

Species Reactivity: 

Human

Antigen Specificity: 

Mouse monoclonal antibody (33 D3) was made against 5-methylcytosine conjugated to ovalbumin. 

Reconstitution and Storage: 

Antibody at a concentration of 1 mg/ml in 10 mM phosphate buffer, 150 mM NaCl, pH 7.4. Store at -20°C. Avoid freeze thaw cycles. 

Key Reference: 

Maki, Gary K., et al., (2007) Biosensors and Bioelectronics (PMID: 17936611 )

Quality Control:

Mouse IgG1 is purified by Protein A affinity chromatography method. 

Suggested starting concentrations are provided. Optimal dilutions should be determined by end-user. Differences in calculated versus apparent molecular weight may be due to post-translational modifications or protein hydrophobicity. For research use only not for diagnostic, human, or veterinary use.

Immunoblot Protocol for Cell Lysates

This protocol is intended to provide additional details for the use of ASB antibodies in immunoblotting. Please consult the product data sheet for the appropriate concentration of primary antibody and any special conditions.

To prepare total cell lysates, spin down cells and re-suspension the pellet in PBS to make the final concentration around 3 x 106 cells / ml. This is a convenient cell density for many cell lines, but adjustments may be necessary for cell types that differ substantially in size and protein content. Prepare cell extracts in appropriate non-reducing or reducing sample buffer as indicated on the experiment or product date sheet. In some cases reducing agents may disrupt the conformation that is recognized by a monoclonal detection antibody. Mix the cell suspension with an equal volume of non-reducing 2X SDS gel sample buffer (6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, and bromophenyl blue) or reducing 2X SDS gel sample buffer [non-reducing buffer plus 20 mM dithithreitol (DTT)]. Sonicate the cells to fragment the DNA using 8-10 bursts of 2-3 seconds each.

1. Load cell extracts and separate proteins on an appropriate percentage SDS polyacrylamide gel (SDS-PAGE) (smaller proteins should be run on a higher percentage gel and higher molecular weight proteins should be run on a lower percentage gel).          

            2. Transfer the separated proteins onto an Immobilon P membrane (Millipore) and incubate the membrane for 1 hour at room temperature or overnight at 2-8 °C in Blocking Solution (1 X PBS, pH 7.4 containing 5% dry milk).              

            3. Wash the membrane at room temperature for 30 minutes with 5 changes of Wash Buffer (1X PBS with 0.1% NP40,).

            4. Incubate the membrane for 2 hours at room temperature or overnight at 2-8 °C in Blotting Buffer (1 X PBS, pH 7.4 containing 5% dry milk unless otherwise indicated) containing primary antibody ( 0.2 to 1ug/ml concentration as indicated on datasheet).      

5. Wash the membrane at room temperature for 30 minutes with 5 changes of Wash Buffer.           

            6. Incubate the membrane at room temperature for 1 hour in Blotting Buffer containing an appropriate secondary reagent such as goat anti rabbit IgG or anti-mouse IgG -HRP at 1:10,000 (Catalog # AviHRP-GAR ).           

            7. Wash the membrane at room temperature for 30 minutes with 5 changes of Wash Buffer.         

            8. Detect with chemiluminescence reagents. (Catalog # Cat. No. AviLight-500).     

            9. Optimal dilutions should be determined by each laboratory for each application.