Optimization of Enzyme Reactions for Screening and Profiling Wild Type EZH2 with the Transcreener EPIGEN Methyltransferase Assay

Discover EZH2 Inhibitors with BellBrook's EZH2 Assay Kits

Numerous studies have shown that histone methyltransferase enhancer of zeste homolog 2 (EZH2) is overexpressed in prostate, breast, bladder, brain, and other tumor types and inhibitors of the enzyme show promise as anti-cancer drugs. EZH2 methyltransferase inhibitors also have shown promise for combating neuropathic pain.

EZH2 is the catalytic component of the polycomb group repressive Complex (PRC2), that selectively methylates histone H3 lysine 27 (H3K27).  Reconstituting EZH2 enzyme reactions in vitro is complex, as it requires an optimal combination of five different protein subunits and a specific acceptor substrate. Please see the materials required section below for more on suggested enzyme and substrate combinations.

Universal Detection of SAH to Measure EZH2 Enzymatic Activity

BellBrook Labs has two assays available to measure EZH2 enzyme activity. Both platforms measure SAH produced by EZH2. The EPIGEN SAH FP Assay uses the coupling enzymes to turn SAH into AMP which is then detected by the Transcreener AMP FP Assay. The AptaFluor SAH TR-FRET Assay uses a split aptamer approach, which upon binding to SAH produces a positive TR-FRET readout. To learn more about the differences in assay technology please go to the methyltransferase assays page to determine what assay might be the best fit for your research.

EPIGEN EZH2 Assay Schematic
AptaFluor EZH2 Assay Schematic

Applications

  • Measure Enzymatic Activity of EZH2
  • Screen Compound Libraries for EZH2 Inhibitors
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling

Features

  • Detection of unlabeled SAH
  • Easy to use, homogenous, one step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method
  • Available in FP or TR-FRET readouts

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

The BellBrook Labs' EZH2 Assay Kits are in a simple, HTS-ready, mix-and-read format. Run your enzyme reaction, add the reagents, and read your plates. The assay is compatible with 96, 384, and 1536-well formats.

Detection of SAH Under EZH2 Initial Velocity

The assay demonstrates linearity when raw data is converted to SAH using a standard curve. The correlation between the enzyme concentration and product formation demonstrates initial velocity conditions and adherence to Michaelis-Menten parameters.

EZH2 Enzyme Titration - EPIGEN Assay

EZH2 Enzyme Titration

Linear Response

EZH2 Under Initial Enzyme Velocity

EZH2 was titrated in 15 μL reactions in the presence of  2 µM SAM and 3 ng/µL Histone H3.3 for 3 hours at 30°C and then quenched with stop reagent followed by addition of detection reagents.  The EC85 concentration was determined to be ~ 5 ng/µL EZH2.

EZH2 Enzyme Titration - AptaFluor Assay

EZH2 Enzyme Titration AptaFluor
EZH2 AptaFluor Assay Stats

Screen for Inhibitors & Perform SAR

Transcreener Assays are designed for screening compound libraries in a high throughput format. Follow up SAR can also be performed using the assay to determine inhibitor potency with ease.

Acquire Accurate Dose Response Measurements - EPIGEN FP Assay

EZH2 Inhibitor Dose Response Curves

Robust Assays Yield Quality Data

The EPIGEN EZH2 Assay Kit was used to determine Z’ (n = 24) in a 384 well format. 5 ng/µL of EZH2 and 3 ng/µL of histone H3.3 were stopped after 3 hours at 30ºC.  A Z’ of 0.71 was achieved.

Far-Red FP & TR-FRET Readouts Validated on Major Multimode Readers

What's Included - Transcreener EPIGEN SAH Assay

What's Included - AptaFluor SAH Assay

What You Will Need

In addition to our EZH2 Assay Kits, Bellbrook Labs also offers the following products to aid in the drug discovery process:

Use Our Assay Selection Tool to Find the Assay That's Right for You!

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