Label-Free Demethylase Assays

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Lysine demethylases (KDMs) play a central role in epigenetic regulation. There are 27 KDMs separated into two classes that function to remove methyl groups from methylated lysine residues on histones, thereby regulating gene expression. Lysine demethylase 1 is a flavin adenine dinucleotide dependent monoamine oxidase that removes mono-/di-methyl groups. The Jumonji C-terminal domain (JmjC) family requires iron (II) and 2-oxoglutarate to remove mono-, di- and tri-methylated states. KDMs are now emerging as valuable target in many therapeutic areas including cancer, neurological and genetic disorders.

What We Offer

SAMDI Tech offers a plug-and-play format for quantitative analysis of demethylase activity using native substrates. Native peptide and protein substrates can be captured and purified onto SAMDI 384 or 1536 biochip arrays for analysis by mass spectrometry. No need for cumbersome fluorescent-labeled substrates that can interfere with activity and lead to increase false positive rates. SAMDI has the capability to monitor all methylated states simultaneously in a single assay.

SAMDI Advantages

  • Fast - Eliminates labels and reduces assay development time
  • Efficient - Multi-analyte detection and 10,000s of samples analyzed per day
  • Quantitative - Measures enzyme kinetics, mechanism of action, and IC50s

Types of Lysine Demethylase Assays

  • Flavin-dependent (LSD1)
  • Iron-dependent (KDM4C, KDM5A, KDM6A, KDM4A, etc.)

Assay Format
SAMDI surface chemistry for purification of peptide analytes prior to mass spectrometry. Demethylase is reacted with peptide substrate and both substrate and product are captured to SAMDI arrays.

SAMDI Mass Spectrometry
SAMDI mass spectrum of a demethylase event where 20% of substrate is converted to product.

Assay Development
SAMDI is quantitative and can measure enzyme kinetics including: Vo vs [Enzyme], enzyme linearity, Km determination, and plate uniformity.