The MLL4 Assay determines MLL4 activity by directly measuring SAH produced during the methylation of Histone H3 Substrate from SAM. The modified riboswitch that selectively binds with SAH discriminates between SAH and related nucleotides, including SAM (which differs by a methyl group). This produces unambiguous detection of SAH in the presence of excess SAM.
The AptaFluor SAH Assay utilizes a far-red, TR-FRET end-point readout for direct detection of SAH in a simple mix-and-read format. SAH produced by the MLL4 enzyme complex drives the assembly of the split aptamer allowing for FRET between a Terbium chelate donor and a DyLight 650 acceptor. The assay provides an excellent tool to screen compound libraries for methyltransferase modulators.
Current non-radioactive SAH detection methods are not sufficiently sensitive to allow detection of many histone methyltransferases (HMTs) using physiological concentrations of SAM. The exquisite affinity and selectivity of the AptaFluor riboswitch overcomes this technical gap, providing screening and profiling of virtually any methyltransferase enzyme with unparalleled sensitivity.
This application note describes methods for using the AptaFluor SAH Methyltransferase Assay to measure MLL4 enzymatic activity in a high-throughput (384-well) format, e.g., for inhibitor screening and/or dose-response measurements.
Run your enzyme reaction, add quenching reagent, add AptaFluor reagents, and read your plates. The MLL4 assay is compatible with 96, 384, and 1536-well formats.
Excellent Z’ under a variety of assay conditions indicate a robust assay. Robust data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Z’ shown here = 0.72.
The assay demonstrates linearity when raw data is converted to SAH using a standard curve. Here, we used 10 μM Histone H3 Substrate and 350 nM SAM. The assay buffer included 50 mM Tris-HCl (pH 8.6), 2 mM MgCl2, 0.02% Triton X-100, and 1 mM DTT. The Enzyme Stop Mix included 1X Enyzme Stop Reagent and 1X SAH Detection Buffer. The SAH Detection Mix included 20 nM P1-Terbium Mix, 40 nM P2-Dylight 650 nM, and 1X SAH Detection Buffer. The enzyme reaction took place for 120 minutes at 30°C. The Detection Mix was added and incubated for 180 minutes at room temperature and read with a Pherastar Plus/FS plate reader.
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| Component | Notes |
|---|---|
| SAM | SAM supplied in the kit should be used for MT enzyme reactions and to create a SAM/SAH standard curve. The MLL4 enzyme reaction contained 350 nM SAM. |
| SAH | SAH is used to create a SAM/SAH standard curve. |
| Enzyme Stop Reagent, 10X | Enzyme Stop Reagent is diluted to 1X in SAH Detection Buffer and used to quench the activity of MT enzyme components. |
| SAH Detection Buffer, 10X | This buffer is used to prepare the SAH Detection Mix and the Enzyme Stop Mix. |
| P1-Biotin | P1-Biotin is a 55-base RNA oligomer with a biotin molecule at the 5' end. It is incubated with TB-Streptavidin to produce P1-Terbium, the donor piece of the SAH aptamer. |
| P2-Dylight 650 | P2-Dylight 650 is an 18-base RNA oligomer with a Dylight 650 molecule at the 3' end. It is the acceptor piece of the SAH aptamer. |
| Tb-Streptavidin | Tb-Streptavidin is used to produce the Tb-labeled (donor) piece of the SAH aptamer. |
| Conjugation Buffer | This buffer is used for the P1-Biotin/Tb-Streptavidin conjugation reaction. |
| Component | Notes |
|---|---|
| MLL4 Enzyme | We have successfully used recombinant human MLL4 Enzyme Complex from Active Motif Cat. #31499. |
| Histone H3 Substrate | We successfully used recombinant Histone H3(1-21) from Anaspec Cat. #AS-61701. The MLL4 enzyme reaction contained 10 μM Histone H3. |
| Enzyme Buffer | 50 mM Tris-HCl (pH 8.6), 2 mM MgCl2, 0.02% Triton X-100, and 1 mM DTT. Enzyme buffer components may be optimized as required. |
| Assay Plates | Assay Plates Can Be Purchased Directly Through BellBrook Labs 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 read TR-FRET is required for the assay. |
| 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 and RNA aptamers. Careful handling and use of ultrapure water eliminates this potential problem. |
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