EZH2 Assay Kits

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.

Transcreener EPIGEN SAH FP Assay - LEARN MORE

AptaFluor SAH TR-FRET Assay - LEARN MORE

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

Linear Response

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

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

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

Instrument	FP Assays	TR-FRET Assays
TriStar²S LB 942	in review	validated
Mithras² LB 943	in review	validated

Cytation™ 5	validated	validated
Cytation™ 3	validated	validated
Cytation™ 1	validated	validated
Synergy™ H1	validated	validated
Synergy™ 2/H4/4	validated	validated
Synergy™ HTX	not capable	not capable
Synergy™ Neo 2	validated	validated

POLARstar^® Omega	validated	validated
FLUOstar^® Omega	not capable	validated
PHERAstar^® FSX	validated	validated
PHERAstar^® Plus/FS	validated	validated
CLARIOstar^® /Plus	validated	validated

Analyst^® GT/HT	validated	validated
Gemini^® XPS/EM	not capable	not capable
SpectraMax^® M2/M2e	not capable	not capable
SpectraMax^® M5/M5e/FlexStation^® 3	not capable	validated
SpectraMax^® Paradigm	validated	contact us

EnVision^®/EnVision^® Xcite	validated	validated

Infinite^® M1000/M1000Pro/Safire2™	validated	validated
Infinite^® M200	not capable	not capable
Infinite^® F500	validated	validated
Infinite^® F200/Ultra^evolution	contact us	contact us
Spark™ 10M	validated	validated

What's Included - Transcreener EPIGEN SAH Assay

Edit
Component	Notes
AMP Antibody	Antibody required for the enzyme is dependent upon the SAM concentration and buffer conditions.
MT Detection Mix, 10X	Coupling enzymes used to convert SAH into AMP. The tracer when displaced, changes in fluorescence can be read in an FP. Included in an assay buffer of 100 mM HEPES and 0.1% Brij-35.
Stop Buffer A, 1X	An acidic reagent that is added directly to the reaction to inhibit MT activity. The reagent is used to run endpoint assays and can be eliminated for kinetic readouts.
SAM	5 mM SAM used for MT enzyme reactions and to create a SAM/SAH standard curve.
SAH	SAH is used to create a SAM/SAH standard curve.

What's Included - AptaFluor SAH Assay

Edit
Component	Notes
Enzyme Stop Reagent, 10X	6% SDS used to quench the activity of MT enzyme Components
SAH Detection Buffer, 10X	Buffer used to prepare the SAH Detection Mix and the Enzyme Stop Mix.
Conjugation Buffer, 1X	Buffer used for the P1-Biotin/Tb-Streptavidin conjugation reaction.
SAM	500 µM SAM used for MT enzyme reactions and to create a SAM/SAH standard curve.
SAH	500 µM SAH is used to create a SAM/SAH standard curve.
P1-Biotin	A 55-base RNA oligomer with a biotin molecule at the 5' end. It is incubated with the Tb-Streptavidin to produce P1-Terbium, the donor piece of the SAH Aptamer.
P2-Dylight 650	A 18-base RNA oligomer with a Dylight 650 molecule at the 3' end. The acceptor pieces of the SAH aptamer.
Tb-Streptavidin	Used to produce the Tb-labeled (donor) piece of the SAH aptamer.

What You Will Need

Edit
Component	Notes
EZH2 Enzyme	We have successfully used recombinant EZH2 complex from Reaction Biology Cat. #HMT-25-114.
Enzyme Buffer	50 mM Tris-HCl (pH 8.5), 5 mM MgCl₂, 0.01% Triton X-100, and 100 mM NaCl buffer, is a suggested starting point.
Substrate	We have found the most success with Histone H3.3. This can be purchased from Reaction Biology Cat. #HMT-11-134.
Assay Plates	Assay Plates Can Be Purchased Directly Through BellBrook Labs FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514. TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513.

BellBrook's EZH2 Assay Kits

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

Cat. No.	Product Description	Price	Add to Cart
3023-B	AptaFluor SAH Methyltransferase Assay (100 Assay, 96 Well)	$375.00	Add to Cart
3023-1K	AptaFluor SAH Methyltransferase Assay (1,000 Assay, 384 Well)	$1,175.00	Add to Cart
3023-10K	AptaFluor SAH Methyltransferase Assay (10,000 Assay, 384 Well)	$6,999.00	Add to Cart
3017-1K	Transcreener EPIGEN Methyltransferase Assay (1,000 Assay, 384 Well)	$975.00	Add to Cart
3017-10K	Transcreener EPIGEN Methyltransferase Assay (10,000 Assay, 384 Well)	$5,850.00	Add to Cart
3017-100K	Transcreener EPIGEN Methyltransferase Assay (10 X 10,000 Assay, 384 Well)	$29,250.00	Add to Cart

Assay Plates for FP

Edit
Cat. No.	Product Description	Price
2233	Corning 96-Well Black Assay Plates (#3686), 3-Pack (200+ Assays)	$40.00	Add to Cart
2229	Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)	$45.00	Add to Cart
2230	Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)	$400.00	Add to Cart

Assay Plates for TR-FRET

Edit
Cat. No.	Product Description	Price
2234	Corning 96-Well White Assay Plates (#3642), 3-Pack (200+ Assays)	$40.00	Add to Cart
2231	Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)	$50.00	Add to Cart
2232	Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)	$450.00	Add to Cart

EZH2 Assay Services

Interested in moving your program forward, but don't want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your EZH2 program.

Lead Discovery Services Include:

Inhibitor Screening – To identify or confirm activity with the target.
Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC₅₀ results.
Residence Time Measurements – Determination of k_off using ‘jump dilution’ enzymatic assay method.
Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.

Contact Us to Learn More

Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

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

Glycosyltransferase Assays

GTPase Assays

Kinase Assays

PDE Assay Kits

EZH2 Assay Resources

Blog Post - EZH2 Methyltransferase Inhibitors Hold Promise for Combating Neuropathic Pain
Blog Post - Preparing for the Epigenevitable
On Demand Webinar - The AptaFluor Methyltransferase Assay: A Homogenous Universal Methyltransferase Assay Based on a Microbial Riboswitch

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

Select Your Assay