Global DNA Hydroxymethylation Assay Kit (5hmc, Colorimetric) (ab233487)
Key features and details
- Detection method: Colorimetric
- Platform: Microplate reader
- Sample type: DNA
Overview
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Product name
Global DNA Hydroxymethylation Assay Kit (5hmc, Colorimetric) -
Detection method
Colorimetric -
Sample type
DNA -
Product overview
The Global DNA Hydroxymethylation Assay Kit (ab233487) is suitable for detecting global DNA hydroxymethylation levels using DNA isolated from any species including mammals, plants, fungi, bacteria, and viruses in a variety of forms including, but not limited to, cultured cells, fresh and frozen tissues, paraffin-embedded tissues, plasma/serum samples, and body fluid samples.
In this assay, DNA is bound to strip-wells that are specifically treated to have a high DNA affinity. The hydroxymethylated fraction of DNA is detected using a 5-hmC mAb-based detection complex in a one-step manner and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer.
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Notes
DNA methylation occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring by DNA methyltransferases, resulting in 5-methylcytosine (5-mC). In somatic cells, 5-mC is found almost exclusively in the context of paired symmetrical methylation of the dinucleotide CpG, whereas in embryonic stem (ES) cells, a substantial amount of 5-mC is also observed in non-CpG contexts. The biological importance of 5-mC as a major epigenetic modification in phenotype and gene expression has been recognized widely.
5-hydroxymethylcytosine (5-hmC), as a sixth DNA base with functions in transcription regulation, has been detected to be abundant in human and mouse brains and embryonic stem (ES) cells. In mammals, it can be generated by the oxidation of 5-mC, a reaction mediated by the ten-eleven translocation (TET) family of 5-mC-hydroxylases.
5-hmC has been demonstrated to be tissue specific, ranging from undetectable levels in cultured cell lines to 0.6% in human brain tissues, and can be as high as 8% of total DNA in some other species. The biological significance of 5-hmC as an important epigenetic modification in phenotype and gene expression has been recently recognized. For example, global decrease in 5-hmC content (DNA hypohydroxymethylation) has been exhibited in nearly all cancers and has been proposed as a molecular marker and therapeutic target in cancer as well.
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Platform
Microplate reader
Properties
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Storage instructions
Please refer to protocols. -
Components 1 x 48 tests 1 x 96 tests 10X Wash Buffer 1 x 14ml 1 x 28ml 5-hmC Antibody, 1000X 1 x 5µl 1 x 10µl 8-Well Assay Strips (with Frame) 1 x 6 units 1 x 12 units Binding solution 1 x 5ml 1 x 10ml Developer Solution 1 x 5ml 1 x 10ml Enhancer Solution, 1000X 1 x 5µl 1 x 10µl Negative Control containing 0% 5-hmC, 50 µg/ml 1 x 50µl 1 x 100µl Positive Control containing 1% 5-hmC, 50 µg/ml 1 x 10µl 1 x 20µl Signal Indicator, 1000X 1 x 5µl 1 x 10µl Stop Solution 1 x 5ml 1 x 10ml
Images
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5-hmC standard curve
An example of an optimal standard curve generated with 5-hmC standard control.
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Quantification of 5-hmC content of DNA from various species.Accurate quantification of 5-hmC content of various DNA samples from different species with the The Global DNA Hydroxymethylation Assay kit (ab233487). The results are closely correlated with those obtained by MS-LC.