Epigenase LSD1 Demethylase Activity/Inhibition Assay Kit (Fluorometric)

The Epigenase™ LSD1 Demethylase Activity/Inhibition Assay Kit (Fluorometric) is a complete set of optimized reagents, designed for an easy and fast fluorometric measurement of LSD1 activity or inhibition. The antibody-based, immunospecific method directly detects LSD1-converted demethylated products, rather than by- products, in a 96 stripwell microplate format. The kit has the following advantages:

• 3 hour procedure in a 96 stripwell microplate format allows for either manual or high throughput analysis.
• Directly measures LSD1 activity via a straightforward detection of LSD1-converted demethylated products, rather than by-products, thus eliminating assay interferences caused by thiol-containing chemicals such as DTT, GSH, and 2-mercaptoethanol.
• Both cell/tissue extracts and purified LSD1 can be used, which allows for the detection of inhibitory effects of LSD1 inhibitors in vivo and in vitro.
• Sensitivity is up to 50 times higher than H₂O₂/formaldehyde release-based LSD1 assays, allowing activity to be fluorometrically detected from as low as 2 ng of purified LSD1 enzyme.
• Demethylated H3-K4 standard is included, allowing specific activity of LSD1 to be quantified. 
• Accurate, reliable, and consistent with extremely low background signals.

About Histone Demethylation
Histone demethylation is the removal of methyl groups in modified histone proteins via histone demethylases. The discovery of histone demethylases demonstrates that histone methylation is not a permanent modification, but rather a more dynamic process. One of the most currently studied enzymes of histone demethylation is lysine specific demethylase 1 (LSD1). LSD1 is found to be pivotal in development and differentiation. For example, this enzyme is required to induce skeletal muscle differentiation, and mutation of drosophila LSD1 results in tissue-specific defects in development through disruption of H3-K4 methylation. LSD1 is also shown to participate in regulation of chromatin remodeling, cell death, and global DNA methylation. More importantly, LSD1 is found to be involved in some pathological processes such as cancer and inflammatory diseases. Detection of activity and inhibition of LSD1 would be important in elucidating mechanisms of epigenetic regulation of gene activation and silencing, and benefit cancer diagnostics and therapeutics.

Principle & Procedure
In this assay, a di-methylated histone H3-K4 LSD1 substrate is stably coated onto microplate wells. Active LSD1 binds to the substrate and removes methyl groups from the substrate. The LSD1-demethylated products can be recognized with a specific antibody. The ratio or amount of demethylated products, which is proportional to enzyme activity, can then be fluorometrically measured by reading the fluoresence in a fluorescent microplate spectrophotometer at 530 excitation and 590 emission. The activity of the LSD1 enzyme is proportional to the fluorescent intensity measured.

 
Fig. 1. Schematic procedure for the Epigenase™ LSD1 Demethylase Activity/Inhibition Assay Kit (Fluorometric).

Fig. 2. Demonstration of high sensitivity of LSD1 activity assay achieved by using recombinant LSD1 with the Epigenase™ LSD1 Demethylase Activity/Inhibition Assay Kit (Flurometric).

Fig. 3. Demonstration of inhibitory effects of an LSD1 inhibitor detected by the Epigenase™ LSD1 Demethylase Activity/Inhibition Assay Kit (Fluorometric). [LSD1 concentration: 200 ng/well].