EpiQuik Global Pan-Methyl Histone H3K9 Quantification Kit (Fluorometric)

The EpiQuik™ Global Pan-Methyl Histone H3-K9 Quantification Kit (Fluorometric) is a convenient package of tools that allows the experimenter to specifically measure global mono-, di-, and tri-methylation of histone H3-K9 fluorometrically using a variety of mammalian cells (human, mouse, etc.) including fresh and frozen tissues, cultured adherent and suspension cells. The kit has the following advantages:

• Quick and efficient procedure, which can be finished within 3 hours.
• Innovative fluorometric assay without the need for radioactivity, electrophoresis, and chromatography.
• Simultaneous quantification of mono-, di-, and tri-methylated H3-K9 with the detection limit as low as 1 ng/well and detection range from 20 ng-5 µg/well of histone extracts.
• The control is conveniently included for the quantification of the amount of mono-, di-, and tri-methylated H3-K9.
• Strip microplate format makes the assay flexible: manual or high throughput.
• Simple, reliable, and consistent assay conditions.

Background Information
Epigenetic activation or inactivation of genes plays a critical role in many important human diseases, especially in cancer. A major mechanism for epigenetic inactivation of the genes is methylation of CpG islands in genome DNA caused by DNA methyltransferases. Histone methyltransferases (HMTs) control or regulate DNA methylation through chromatin-dependent transcription repression or activation. HMTs transfer 1-3 methyl groups from S-adenosyl-Lmethionine to the lysine and arginine residues of histone proteins. ESET, G9a, SUV39-h1, SUV39- h2, SETDB1, Dim-5, and Eu-HMTase are histone methyltransferases that catalyze methylation of histone H3 at lysine 9 (H3-K9). In mammalian cells, mono-methyl H3-K9 are enriched in certain euchromatic domains, which have been postulated to be transcriptionally silent. H3-K9 di- and trimethylation mediates heterochromatin formation by forming a binding site for HP1 and also participates in silencing gene expression at euchromatic sites. Increased global H3-K9 methylation is also found to be involved in some pathological processes such as cancer progression. The patterns of global H3-K9 methylation can also be changed by inhibition or activation of HMTs. Thus, quantitative detection of the patterns of global histone H3-K9 methylation would provide useful information for better understanding epigenetic regulation of gene activation/silencing, and for developing HMT-targeted drugs. The EpiQuik™ Global Pan-Methyl Histone H3-K9 Quantification Kit (Fluorometric) provides a tool for measuring global mono-, di-, and trimethylation of histone H3-K9.

Principle & Procedure
This kit is designed for simultaneously measuring global histone H3-K9 mono-, di-, and tri-methylation. In an assay with this kit, the methylated histone H3 at lysine 9 is captured to the strip wells coated with antibodies specifically for mono-, di-, and tri-methyl H3-K9. The captured mono-, di-, and tri-methylated histone H3-K9 can then be detected with a labeled detection antibody, followed by a fluorescent development reagent. The ratio of mono-, di-, and tri-methylated H3-K9 is proportional to the intensity of fluorescence. The absolute amount of the methylated H3-K9 can be quantitated by comparing to the standard control.

Starting Materials
Input material should be purified histone extracts. In general, the input amount should be from 50 ng to 200 ng per well of histone extracts.