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Poster: AptaFluor® SAH: A Homogenous, Universal Methyltransferase Assay Based on a Microbial Riboswitch

MLL4 Assay - An AptaFluor SAH Assay Application

The AptaFluor SAH Assay measures MLL4 (Mixed-Lineage Leukemia 4 or KMT2D) activity in a high-throughput format for inhibitor screening/dose-response measurements. AptaFluor uses a naturally occurring SAH-sensing RNA aptamer to directly detect SAH produced during the methylation of substrates. The exquisite affinity and selectivity of the riboswitch enables profiling methyltransferases with unparalleled sensitivity.

The kit comes complete with the detection reagents required to measure activity. MLL4 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme. This assay is designed to be used with purified enzyme preparations. AptaFluor assays are not validated with cell lysates, blood, serum, or other biological samples.

Application Note - Mixed-Lineage Leukemia 4 Activity with the AptaFluor SAH Methyltransferase Assay

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.

Application Note Preview Image

How Does The Assay Work?

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. 

Measure MLL4 Activity with a TR-FRET Readout

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

Schematic of MLL4 Assay

The Most Sensitive HTS Methyltransferase Assay

The AptaFluor Assay is an ultra-sensitive assay that allows a LLD of 0.6 nM SAH. This drastically reduces enzyme usage and allows the assay to be run at or below KM for SAM.

Ulta Sensitive Methyltransferase Assay
Data Table Demonstrating AptaFluor Sensitivity

Easy-to-Use, Mix-&-Read, HTS-Ready

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.

AptaFluor Methyltransferase Assay Protocol Outline

Applications

  • Measure enzymatic activity of MLL4
  • Screen Compound Libraries for MLL4 Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling

Features

  • Use SAM concentrations as low as 100 nM and as high as 5 μM
  • Direct detection of SAH
  • Simple, mix-and-read format ideal for HTS.
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method

Robust Assay Yields Quality Data

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.

MLL4 Z' Data

Detection of SAH Under MLL4 Initial Velocity Conditions

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.

MLL4 Enzyme Titration

MLL4 Assay Enzyme Titration

Linear Response

MLL4 Linear Response Curve

MLL4 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 MLL4 program.

Assay Development Services

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 koff 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.

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

SupplierInstrumentTR-FRET Assays
TriStar2S LB 942Click Me ArrowValidated
Tristar 5In Review
Mithras2 LB 943Click Me ArrowValidated
CytationTM 5Validated
CytationTM 3Validated
CytationTM 1Validated
SynergyTM H1Validated
SynergyTM 2/H4/4Click Me ArrowValidated
SynergyTM HTXNot Capable
SynergyTM Neo 2Validated
POLARstar® OmegaClick Me ArrowValidated
FLUOstar® OmegaClick Me ArrowValidated
PHERAstar® FSXClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidated
SenseIn Review
Analyst® GT/HTClick Me ArrowValidated
Gemini® XPS/EMNot Capable
SpectraMax® M2/M2eNot Capable
SpectraMax® M5/M5e/FlexStation® 3Click Me ArrowValidated
SpectraMax® ParadigmClick Me ArrowContact Us
SpectraMax® iD3/iD5Click Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidated
Infinite® M200Not Capable
Infinite® F500Click Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidated

What's Included

What You Will Need

AptaFluor SAH Methyltransferase Assay Kits - For MLL4 Activity Assays

* For custom or bulk orders (over 10,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

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