EpiQuik Global Pan-Methyl Histone H4K20 Quantification Kit (Fluorometric)

The EpiQuik Global Pan-Methyl Histone H4K20 Quantification Kit (Fluorometric) is a convenient package of tools that allows the experimenter to specifically measure global mono-, di- and tri-methylation of histone H4-K20 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 2.5 hours.
• Innovative fluorometric assay without the need for radioactivity, electrophoresis, or chromatography.
• Simultaneously quantify mono-, di-, and tri-methylated H4-K20 with the detection limit as low as 1 ng/well, and detection range from 10 ng-2 µg/well of histone extracts.
• The control is conveniently included for the quantification of pan-methylated H4-K20.
• 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 play 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 transcriptional repression or activation. HMTs transfer 1-3 methyl groups from S-adenosyl-Lmethionine to the lysine and arginine residues of histone proteins. PR-SET7, SET9, SUV4.20h, and ASH1 are histone methyltransferases that catalyze methylation of histone H4 at lysine 20 (H4-K20) in mammalian cells. H4-K20 mono-methylation is involved in the maintenance of proper higher order structure of DNA and is consequently essential for chromosome condensation, as well as functioning in gene silencing. H4-K20 di-methylation has been described as a repressive chromatin domain and is involved in DNA damage response. H4-K20 tri-methylation acts as a passive feature or structure determinant for chromatin degradation and release and is an epigenetic marker of early apoptosis. Tri-methylation of H4-K20 is also considered to be a common hallmark of human cancer. The global H4-K20 methylation can be changed by inhibition or activation of HMTs. Thus, quantitative detection of global methyl histone H4-K20 would provide useful information for better understanding epigenetic regulation of gene activation/repression, as well as for developing HMT-targeted drugs. The EpiQuik™ Global Pan-Methyl Histone H4-K20 Quantification Kit (Fluorometric) provides a tool for measuring global mono-, di-, and tri-methylation of histone H4-K20.

Principle & Procedure
This kit is designed for simultaneously measuring mono-, di-, and tri-methylation of histone H4-K20. In an assay with this kit, the mono-, di-, and tri-methylated histone H4 at lysine 20 is captured to the strip wells coated with an anti-mono-, di-, and tri-methyl H4-K20 antibody. The captured methylated histone H4-K20 can then be detected with a detection antibody followed by a fluorescent development reagent. The ratio of mono-, di-, and tri-methylated H4-K20 is proportional to the intensity of fluorescence. The absolute amount of mono-, di-, and tri-methylated H4-K20 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.