The Epigenase™ PRMT Methyltransferase (Type II-Specific) Activity/Inhibition Assay Kit (Colorimetric) is a complete set of optimized buffers and reagents for measuring the activity or inhibition of total type II PRMT using nuclear extracts or purified enzymes such as PRMT5 and PRMT7 from a broad range of species such as mammals, plants, fungi, and bacteria, in a variety of forms including cultured cells and fresh tissues. This kit has the following advantages:
- 3 hour colorimetric procedure in a 96 stripwell microplate format allows for either manual or high throughput analysis.
- Directly measures type II PRMT activity via a straightforward detection of PRMT-converted methylated products.
- Both cell/tissue extracts and purified type II PRMT can be used, which allows for the detection of inhibitory effects of PRMT5 or PRMT7 inhibitors in vivo and in vitro.
- Sensitive detection limit can be as low as 5 ng of purified PRMT5 enzyme.
- Methylated H4-Arg3 standard is included, allowing for specific activity of type II PRMT to be quantified.
- Accurate, reliable, and consistent with extremely low background signals.
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). 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 (CARM1, PRMT1, PRMT2, PRMT3, PRMT6, and PRMT8) and type II (PRMT5 and PRMT7).
Type II PRMTs are found to be strongly implicated in diseases like cancer. Detection of activity and inhibition of type II PRMTs would be important in elucidating mechanisms of epigenetic regulation of gene activation and silencing, as well as benefiting cancer diagnostics and therapeutics.
Principle & Procedure
In this assay, a type II PRMT substrate is stably coated onto microplate wells. Active PRMT5 or PRMT7 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 II PRMT enzymes is in turn proportional to the optical density intensity measured.
Starting Materials & Input Amount
Input materials can be nuclear extracts or purified PRMT5/PRMT7 enzymes. 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.
Fig. 1. Schematic procedure of the Epigenase™ PRMT Methyltransferase (Type II-Specific) Activity/Inhibition Assay Kit (Colorimetric).
Fig. 2. Demonstration of high sensitivity of the type II PRMT activity assay achieved by using recombinant PRMT5 with the Epigenase PRMT (Type II-Specific) Meethyltransferase Activity/Inhibition Assay Kit (Colorimetric).