The identification of histone demethylases (HDMT) catalyzing the removal of methyl groups from modified histone proteins has indicated that demethylation events play an important role in histone modification dynamics. Just like all other histone modifying enzymes, histone demethylases play an important role in development and gene regulation, and many of them require a cofactor like Fe(II), FAD, or α-ketoglutarate. HDMTs display a high level of substrate specificity to their target lysine or arginine, as well as high sensitivity to the specific type of methylation to be recognized and demethylated. Currently, the most commonly studied histone demethylases are LSD1 and JARIDs, where LSD1 can eliminate mono- and di-methylation from H3-K4 by using an amine oxidase reaction. The role of histone demethylases in the chromatin regulation has provided new insight for understanding the epigenetic landscape of the chromatin environment of cancer cells, thus making it necessary to better understand the activity or inhibition of HDMTs and clarify their potential mechanisms in cancer therapy.
As a proficient contract research organization, Creative BioMart is able to provide customers with low-cost high throughput screening assays on HDMT based on chemiluminescent or fluorescent protocol in an efficient manner. A group of well-trained, knowledgeable medicinal biochemists at Creative BioMart are dedicated to working with you to stringently identify the optimal inhibitors against your target HDMT from a library of compounds within the shortest timeline. Furthermore, a unique and comprehensive assembly of HDMTs, substrates, assay kits, and inhibitors/activators provided by Creative BioMart make our company a trusted partner to offer innovative research techniques for biotechnology industry around the world.
Procedure of Histone Demethylases Screening at Creative BioMart
a. Chemiluminescent Method
Histone demethylase assays at Creative BioMart is based on highly specific antibodies recognizing demethylated products. A sample from clients is incubated with a targeted HDMT and a biotinylated substrate. Subsequently, acceptor beads, primary antibodies and donor beads are added to the reaction system. The Alpha-counts is finally read.
Figure 1. General procedure of chemiluminescent method for HDMT screening assay
b. Fluorometrical Method
Fluorometrical method used by Creative BioMart allows a convenient measure of histone demethylase inhibition, where the inhibitors directly interact with HDMT or block the binding of the enzyme to its substrate. In the assay, HDMT is incubated with the unique substrate and inhibitor in the strip wells, where the binding of enzyme with substrate produces hydrogen peroxide. Then the fluorescent developing solution is added to react with the produced hydrogen peroxide, thus obtaining the highly fluorescent oxidation products. The intensity of fluorescence from oxidation products is proportional to enzyme activity. Therefore, as enzyme activity decreases by inhibition, the fluorescent signal decreases.
Figure 2. Schematic procedure of fluorometrical method for HDMT screening assay
Advantages of Our Service
Creative BioMart supplies compounds screening and profiling against HDMTs for your epigenetic drug discovery through our top-ranking equipment. A variety of HDMT targets are accessible to meet all of your research demands and help you process your research. Our highly experienced team is willing to give you advice for the best screening approach depending on the nature of the project and the resources availability. For more detailed information, please feel free to contact us or directly sent us an inquiry.
Reference
1. Gale, M.; Yan, Q. High-throughput screening to identify inhibitors of lysine demethylases. Epigenomics. 2015, 7(1): 57–65.
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