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Specifications

  • Assay Type Activity Assay
  • Sample Types Lysates, Purified Enzyme Systems
  • Assay Duration 1+ hour enzyme reaction, 30 minute assay incubation
  • Samples/Plate 40 in Duplicate
  • Readout Fluorescent, 510 nm emission / 450 nm excitation
  • Standard Curve
  • Description

    Assay Principle: 

    The Histone Demethylase (HDM) Fluorescent Activity Kit quantitatively measures HDM activity in lysates and purified enzyme systems. The Histone Demethylase (HDM) Fluorescent Activity Kit is an Activity Assay with a run time of 30 minutes. The kit is validated for LSD1 and JMJD2A Histone Demethylases (HDMs). Please read the complete kit insert for more information before performing this assay.

    Use our provided Formaldehyde Standard to generate a standard curve for the assay. Pipette the standards or diluted samples into a black microtiter plate. Allow the demethylase reaction to occur until your desired stop point. Add DetectX® Formaldehyde Detection Reagent to each well tapping the plate to ensure sufficient mixing of reagents. Then incubate the mixture covered at 37°C for 30 minutes. The fluorescent generating reaction occurs between the Formaldehyde Detection Reagent and the Formaldehyde generated from the demethylase reaction within the samples. 

    After the 30-minute incubation, use a plate reader to detect and record the generated fluorescent signal at 510nm. Use the intensity and the standard curve to calculate the HDM activity in the samples.

    Background:

    Histone demethylases (HDMs) catalyze the site-specific demethylation of methyl-lysine residues in histones to dynamically regulate chromatin structure, gene expression, and potentially other genomic functions. At present, there are two known classes of HDMs: the flavin adenine nucleotide (FAD)-dependent Lysine Specific Demethylase 1 (LSD1) family and the Fe(II)-dependent Jumonji C (JmjC) family. Although they employ different cofactors and catalytic mechanisms, both classes produce Formaldehyde as a byproduct of the demethylation reaction. HDMs have proven challenging to assay quantitatively owing to their relatively low turnover numbers. For this reason, understanding of HDMs’ kinetic properties, substrate specificities, and reaction mechanisms is limited.