The EpiQuik™ Global Tri-Methyl Histone H3-K4 Quantification Kit (Fluorometric) is a convenient package of tools that allows the experimenter to specifically measure global tri-methylation of histone H3-K4 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, and chromatography.
- Specifically captures tri-methyl H3-K4 with the detection limit as low as 0.4 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 tri-methylated H3-K4.
- Strip microplate format makes the assay flexible: manual or high throughput.
- Simple, reliable, and consistent assay conditions.
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 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. SET1, SET7/9, Ash1, ALL-1, MLL, ALR, Trx, and SMYD3, are histone methyltransferases that catalyze methylation of histone H3 at lysine 4 (H3-K4) in mammalian cells. H3-K4 tri-methylation has been viewed as a signature mark of highly transcribed genes, which is placed exclusively in the 5’- region downstream of the promoter. Increased global H3-K4 tri-methylation is also found to be involved in some pathological processes such as cancer progression. The global H3-K4 tri-methylation can also be changed by inhibition or activation of HMTs. Thus, quantitative detection of global tri-methyl histone H3-K4 would provide useful information for better understanding epigenetic regulation of gene activation and for developing HMT-targeted drugs. The EpiQuik™ Global Tri-Methyl Histone H3-K4 Quantification Kit (Fluorometric) provides a tool for measuring global tri-methylation of histone H3-K4.
Principle & Procedure
This kit is designed for measuring global histone H3-K4 tri-methylation. In an assay with this kit, the tri-methylated histone H3 at lysine 4 is captured to the strip wells coated with an anti-trimethyl H3-K4 antibody. The captured tri-methylated histone H3-K4 can then be detected with a labeled detection antibody, followed by a fluorescent development reagent. The ratio of tri-methylated H3-K4 is proportional to the intensity of fluorescence. The absolute amount of tri-methylated H3-K4 can be quantitated by comparing to the standard control.
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.