Epigenase Type I PRMT Methyltransferase Activity/Inhibition Assay Kit (Colorimetric) | P-3098

The Epigenase™ Type I PRMT Methyltransferase Activity/Inhibition Assay Kit (Colorimetric) is a complete set of essential components for measuring activity or inhibition of total type I PRMT using nuclear extracts or purified enzymes such as PRMT1 to PRMT 4 from a broad range of species such as mammals, plants, fungi, and bacteria, in a variety of forms including cultured cells and fresh tissues.

The Epigenase™ Type I PRMT Methyltransferase Activity/Inhibition Assay Kit (Colorimetric) DataSheet

The kit has the following advantages and features:

• 3-hour colorimetric procedure in a 96 stripwell microplate format allows for either manual or high throughput analysis
• Directly measures type I PRMT activity via a straightforward detection of PRMT-converted methylated products
• Both cell/tissue extracts and purified type I PRMT can be used, which allows for the detection of inhibitory effects of type I PRMT inhibitors in vivo and in vitro
• Sensitive detection limit can be as low as 10 ng of purified PRMT6 enzymes
• Methylated H4-Arg3 standard is included, allowing for specific activity of type I PRMT to be quantified
• Accurate, reliable, and consistent with extremely low background signals

Background
Arginine histone methylation is one of the many important epigenetic marks, and is essential for the regulation of multiple cellular processes. Arginine methylation of histones H3 (Arg2, 17, 26) and H4 (Arg3) promotes transcriptional activation and is mediated by a family of protein arginine methyltransferases (PRMTs). There are 9 types of PRMTs found in humans but only 7 members are reported to methylate histones. They can mediate mono or dimethylation of arginine residues. These enzymes use S-adenosyl-methionine (SAM) as a methyl donor and transfer it to the guanidinium side chain of arginine. Based on the position of methyl group addition, the PRMTs can be classified into type I (PRMT1, PRMT2, PRMT3, CARM1/PRMT4, PRMT6, and PRMT8) and type II (PRMT5 and PRMT9).

Principle & Procedure
In this assay, a type I PRMT substrate is stably coated onto microplate wells. Active type 1 PRMTs such as PRMT1-4 or PRMT6 and PRMT 8 bind to the substrate and transfer a methyl group from Adomet to methylate the substrate. The methylated products can be recognized with a specific antibody. The ratio or amount of methylated products, which is proportional to enzyme activity, can then be colorimetrically measured by reading the absorbance in a microplate spectrophometer at a wavelength of 450 nm. The activity of the type I PRMT enzymes is, in turn, proportional to the optical density intensity measured.

Starting Material
Input materials can be nuclear extracts or purified type I enzymes such as PRMT1 to PRMT4. The amount of nuclear extracts for each assay can be 1 µg to 20 µg with an optimal range of 5 to 10 µg. The amount of purified enzymes can be 10 ng to 500 ng, depending on the purity and catalytic activity of the enzymes.