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Chemical/ Small Molecule corresponding to 5-methylcytosine (5-mC).
Storage in frost-free freezers is not recommended. Should this product contain a precipitate microcentrifugation before use.While older lots have performed well in ICC, we have received inconsistent results with the latest lots. Unfortunately, we can no longer guarantee this antibody for use in ICC.
Our Abpromise guarantee covers the use of ab10805 in the following tested applications.
|IHC-P||Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|MeDIP||Use at an assay dependent concentration. PubMed: 17845077Used 5ul in 200ul reaction with 2ug digested genomic DNA from Drosophila.|
|ChIP||Use at an assay dependent concentration.|
|IP||Use at an assay dependent concentration.|
|Dot blot||Use at an assay dependent concentration. PubMed: 24386123|
|Flow Cyt||Use at an assay dependent concentration.
Use 10ul of working dilution to label 1000000 cells in 100ul. (see Habib, M. et al. (1999)).
|IHC-Fr||Use at an assay dependent concentration.|
ab10805 staining 5-Methyl Cytidine in pig embryo (15 to 17 days) tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent fixation in paraformaldehyde, heat mediated antigen retrieval in Tris-EDTA buffer, permeabilization in Triton X-100 and blocking in 2% BSA for 10 minutes at 25°C. The primary antibody was diluted, 1/100 (PBS + 2% BSA) and incubated with sample for 1 hour at 25°C. An Alexa Fluor® 488 conjugated donkey polyclonal to mouse at 1/250 dilution, was used as secondary.
Native ChIP analysis using unpurified ab10805 binding 5-methylcytosine (5-mC) in nuclear cell lysate derived from mouse embryonic stem cells. Samples were incubated with primary antibody (2µg/µg chromatin) for 16 hours at 4°C in a Glycerol IP buffer. Protein binding was detected using real-time PCR.
Positive control: Magoh2 in HMT KO cells.
Negative Control: Magoh2 in WT cells.
Dot blot carried out using ab10805 (left blot), showing specificity to 5-methylcytosine. Indicated amounts (20–5 ng) of oligonucleotides containing either 5mC (50%) or 5hmC (20 %) were spotted on positive charged nylon membrane and detected either with 5mC (ab10805) or 5hmC antibody.