Bellbrook Labs has two assays available to measure EZH2 enzyme activity:
The EZH2 Assay using Aptafluor’s SAH Assay determines EZH2 activity by directly measuring SAH (s-adenosylhomocysteine) produced in methylation of various substrates. This EZH2 Assay uses a natural occurring aptamer, or riboswitch, that selectively binds SAH. The exquisite affinity & selectivity of the riboswitch enables screening & profiling of methyltransferases with unparalleled sensitivity.
Transcreener’s EPIGEN Assay determines EZH2 activity via coupling enzymes that convert SAH to AMP. Transcreener’s AMP Assay measures the produced AMP. This technology uses a simple but highly effective method consisting of an antibody selective to AMP and a far-red fluorescent tracer. AMP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.
The EZH2 Assay is available with FP (Transcreener EPIGEN Assay) or TR-FRET (AptaFluor SAH Assay). It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagents, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.
Run your enzyme reaction, add detection reagents, and measure. The EZH2 Assay is compatible with 96, 384, and 1536-well formats.
EPIGEN EZH2 Assay used to determine Z’ (n = 24). 5 ng/µL of EZH2 and 3 ng/µL of histone H3.3 were stopped after 3 hours at 30ºC. Z’ = 0.71.
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 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.
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.
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| 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. |
| 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. |
| 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. |
| Plate Reader | A multi-detection microplate reader configured to measure the output of the tracer is required. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates. |
| Ultrapure Water | Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem. |
Histone methyltransferase enhancer of zeste homolog 2 (EZH2) is the catalytic component of 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.
Numerous studies have shown that EZH2 is overexpressed in prostate, breast, bladder, and brain tumors. Inhibitors of the enzyme show promise as immune-oncology drugs. EZH2 methyltransferase inhibitors also have shown promise for combating neuropathic pain. BellBrook’s EZH2 Assay is an excellent tool for researchers examining the therapeutic effects of EZH2.
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BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400
info@bellbrooklabs.com
