Sheep Anti 5-Methylcytosine (5-MeC)specifically recognizes the methylated form of cytosine (C), an epigenetic modification catalyzed by DNA methyltransferases resulting in the attachment of a methyl group to carbon 5. This modification alters the structure of cytosine but not its base-pairing properties. The timing and function of cytosine methylation varies amongst species with roles in such processing events as transcription and recombination. For example, in mammals, methylation occuring during early development and emrbyogensis functions in X chromosome inactivation and gene imprinting, wherease in plants, DNA methylation occurs later and affects genomic stability through silencing of transposable elements (Heet al.2011).
The deamination of 5-MeC results in the formation of thymine (T) instead of uracil. Since thymine is not recognized by DNA repair enzymes, cytosine to thymine conversions can cause transition mutations. The most frequent mutations in human genetic diseases are those involving C to T transitions occurring at CG sites (Morganet al.2004). Another type of mutation involving 5-MeC involves the formation of pyrimidine dimers. Upon exposure to sunlight, pyrimidine dimers form preferentially at dipyrimidine sequences containing 5-MeC, a mutation frequently seen in human skin cancers (Pfeiferet al.2005).
Sheep Anti-5-MeC is reported as suitable for use in immunocytochemistry.
Species Cross-Reactivity
Target Species
Cross Reactivity
Rat
Yes
Application
This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visitwww.abdserotec.com/protocols.
Application Name
Yes
No
Not Determined
Suggested Dilution
ELISA
Flow Cytometry
Immunofluorescence
Immunohistology - Frozen(1)
10ug/ml -
Immunohistology - Paraffin(2)
10ug/ml -
Immunoprecipitation
25 - 100ug/ml
Western Blotting
1:1000 -
(1)
Using a fixation step in cold methanol for 30 minutes followed by immersion in 0.007 M NaOH for 10-15 seconds allows staining with the simultaneous detection of nuclear cytoplasmic and membrane components, as well as preservation of morphological detail.
(2)
* Using a fixation step in cold methanol for 30 minutes followed by immersion in 0.007 M NaOH for 10-15 seconds allows staining with the simultaneous detection of nuclear cytoplasmic and membrane components, as well as preservation of morphological detail.
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.
Storage
Store at -20oC only.
Storage in frost-free freezers is not recommended.
This product should be stored undiluted. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.
Shelf Life
18 months from date of despatch.
Antiserum Preparation
Antiserum to 5-MeC was raised by repeated immunisation of sheep with highly purified antigen. Purified IgG was prepared by affinity chromatography.
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