The EpiQuik™ Histone Methyltransferase Activity/Inhibition Assay Kit (H3-K4) is a convenient set of tools that allows the experimenter to measure the activity or inhibition of individual histone methyltransferase that specifically target histone H3 at lysine 4 (H3-K4). The kit is ready-to-use and provides all the essential components needed to carry out a successful HMT activity/inhibition experiment without the need for radioactivity or any special equipment.

WHY CHOOSE THE EPIQUIK™ HISTONE METHYLTRANSFERASE ACTIVITY/INHIBITION ASSAY KIT (H3-K4)?

• Very rapid procedure, which can be completed within 3 hours.
• Safe and innovative colorimetric assay without radioactivity, extraction, and chromatography.
• Specific measurement of activity/inhibition of H3-K4 histone methyltransferases.
• Strip microplate format makes the assay flexible: manual or high throughput analysis.
• Extremely simple, reliable, and consistent assay conditions.

The EpiQuik™ Histone Methyltransferase Activity/Inhibition Assay Kit (H3-K4) is designed for measuring HMTs that specifically target histone H3 at lysine 4. In an assay with this kit, the histone substrate is stably captured on the strip wells. HMT enzymes transfer a methyl group to histone H3 substrate from Adomet to methylate the substrate at lysine 4. The methylated histone H3-K4 can be recognized with a high-affinity antibody. The ratio or amount of methylated H3-K4, which is directly proportional to enzyme activity, can be quantified through a HRP conjugated secondary antibody-color development system. The HMT activity is then calculated based on the amount of methylated H3-K4 converted by the HMTs.

SCHEMATIC PROCEDURE:
	Nuclear extracts were prepared from MCF-7 cells using the EpiQuik Nuclear Extraction Kit and H3-K4 specific histone methyltrasferase activity (SET7/9) was measured.

HM1 (10X Wash Buffer)
HM2 (Histone Assay Buffer)
HM3 (Adomet)*
HM4 (Biotinylated Substrate, 25 µg/ml)*
HM5 (HMT Standard, 10 µg/ml)*
HM6 (Capture Antibody, 100 µg/ml)*
HM7 (Detection Antibody, 200 µg/ml)*
HM8 (Developing Solution)
HM9 (Stop Solution)
Control Enzyme (100 µg/ml)*
8-Well Assay Strip (with Frame)
User Guide

* For maximum recovery of the products, centrifuge the original vial after thawing prior to opening the cap.

1. What is the principle of the assay in kit P-3002?
The histone H3 substrate is first captured to the assay wells. H3-K4 site-specific enzymes transfer the methyl group from Adomet to histone H3 at lysine 4 and methylate this lysine site. The methylated H3-K4 can then be recognized with an anti-dimethyl H3-K4 antibody. The ratio or amount of methylated H3-K4, which is proportional to enzyme activity, can be colorimetrically quantified through an ELISA-like reaction.

2. Is kit P-3002 suitable for rat samples?
Yes.

3. Is the assay done in 8-well strips transferable to an older plate reader?
The solution in the 8-well strips can be transferred to the 96-well plate for reading. Also the 8-well strips can be put in the 96-well plate size frame (included in the kit) and directly read with the plate reader.

4. Is it HMT enzyme that is used to obtain the standard curve in this kit?
In the P-3002 kit, histone methyltransferase (HMT)-converted products are used for standard curve and HMT is only used as the positive control.

[User Guide]* *Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

[Material Safety Data Sheet]

Li, Y. et. al. (January 2013). Epigenetic Regulation of Multiple Tumor-Related Genes Leads to Suppression of Breast Tumorigenesis by Dietary Genistein. PLoS One. 8(1):e54369. PubMed Abstract

Zou, Y. et. al. (July 2012). Effect of phenylhexyl isothiocyanate on aberrant histone H3 methylation in primary human acute leukemia. J Hematol Oncol. 5:36. PubMed Abstract

Batra, V. et. al. (March 2012). Interaction between γ-radiation and dietary folate starvation metabolically reprograms global hepatic histone H3 methylation at lysine 4 and lysine 27 residues. Food Chem Toxicol. 50(3-4):464-72. PubMed Abstract

Rugg-Gunn, PJ. et. al. (June 2010). Distinct histone modifications in stem cell lines and tissue lineages from the early mouse embryo. Proc Natl Acad Sci USA. 107(24):10783-90. PubMed Abstract Full PDF Article

Sebastian, S. et. al. (March 2009). MLL5, a trithorax homolog, indirectly regulates H3K4 methylation, represses cyclin A2 expression, and promotes myogenic differentiation. Proceedings of the National Academy of Sciences. 160(12): 4719-24.