Assay Principle:

The DetectX® Histone Demethylase (HDM) Fluorescent Activity Kit provides an efficient and precise method to measure HDM activity in lysates and purified enzyme systems. The kit has a run time of approximately 1.5 hours and is specifically validated for LSD1 and JMJD2A Histone Demethylases. Read the complete kit insert for detailed instructions. A Formaldehyde Standard is included to establish an accurate standard curve.

Protocol Summary:

• Introduce standards or diluted samples into the black microtiter plate.
• Allow the demethylase reaction to proceed to the desired stop point.
• Add DetectX® Formaldehyde Detection Reagent to each well, ensuring thorough mixing.
• Incubate at 37°C for 30 minutes. During incubation a fluorescent reaction occurs between the Detection Reagent and the Formaldehyde produced in the demethylase reaction.
• Use a plate reader to measure the fluorescent signal at 510nm and calculate HDM activity based on the standard curve.

Background:

Histone demethylases (HDMs) are enzymes that remove methyl groups from lysine residues on histones. They play a major role in the dynamic regulation of chromatin structure and gene expression. Currently, scientist recognize two main classes of HDMs: the FAD-dependent Lysine Specific Demethylase 1 (LSD1) family and the Fe(II)-dependent Jumonji C (JmjC) family. Despite employing distinct cofactors and mechanisms, both classes generate Formaldehyde as a byproduct of demethylation.

HDMs are critical in understanding epigenetic regulation but have historically been challenging to assay quantitatively due to low turnover numbers. This limitation has restricted comprehensive knowledge about their kinetic properties, substrate specificities, and reaction mechanisms. The DetectX® Histone Demethylase (HDM) Fluorescent Activity Kit addresses these challenges by offering a sensitive and straightforward method to quantitatively measure HDM activity. This capability is essential for advancing research in epigenetics, including studies on chromatin remodeling, gene regulation, and potential genomic functions influenced by histone modification.

DetectX® Histone Demethylase (HDM) Fluorescent Activity Kit’s sensitive detection system provides valuable insights into the enzymatic activity of HDMs, supporting research efforts to understand the complexities of epigenetic regulation and its implications in health and disease.