The EpiQuik™ Global Di-Methyl Histone H4R3 Quantification Kit (Colorimetric) is a complete set of optimized buffers and reagents to colorimetrically quantify global histone H4 arginine 3 di-methylation from a broad range of species such as mammals, plants, fungi, and bacteria, in a variety of forms including cultured cells and fresh tissues. Histone extracts can be prepared with your own successful method, or prepared with the EpiQuik™ Total Histone Extraction Kit. This kit has the following advantages:
- Quick and efficient procedure, which can be finished within 3.5 hours.
- Innovative colorimetric assay without the need for radioactivity, electrophoresis, or chromatography.
- Specifically captures symmetric di-methylated H4R3 with the detection limit as low as 0.5 ng/well and detection range from 100 ng to 2 µg/well of histone extracts.
- An assay control is conveniently included for the quantification of di-methylated H4R3.
- Strip microplate format makes the assay flexible: manual or high throughput.
- Simple, reliable, and consistent assay conditions.
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 di-methylation 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). Symmetric di-methylation of histone H4 arg3 (H4R3) is catalyzed by type II PRMTs, which are found to be strongly implicated in diseases like cancer. For example, PRMT5 plays a role in the repression of certain tumor suppressor genes such as RB tumor suppressors while PRMT7 overexpression is observed in breast cancer. The global H4R3 di-methylation can be changed by inhibition or activation of type II PRMTs. Therefore, quantitative detection of global symmetric di-methyl histone H4R3 would provide useful information for better understanding epigenetic regulation of gene activation and silencing, as well as for developing PRMT-targeted drugs.
Principle & Procedure
In this assay, the histone proteins are stably spotted on the strip wells. The di-methyl histone H4R3 can be recognized with a high-affinity antibody and detected with a detection antibody, followed by a color development reagent. The ratio of di-methylated H4R3 is proportional to the intensity of absorbance. The absolute amount of di-methylated H4R3 can be quantitated by comparing to the standard control.
Input materials can be histone extracts or nuclear extracts. The amount of histone extracts for each assay can be 0.1 µg to 2 µg with an optimal range of 0.5 to 1 µg.
WB (10X Wash Buffer)
HB (Histone Buffer)
BB (Blocking Buffer)
MER3 (Capture Antibody, 1000X)
RDA (Detection Antibody, 2000X)
ES (Enhancer Solution)
DS (Developer Solution)
SS (Stop Solution)
DMC Di-Methyl H4R3 control (50 µg/ml)
8-Well Assay Strips (With Frame)
Adhesive Covering Film