Poster: Case Study for PRMT5, MLL4, METTL3/14 and NSP14 Using the AptaFluor® Assay

AptaFluor SAH Methyltransferase Assay

AptaFluor SAH Methyltransferase Assay directly measures SAH produced by methyltransferases during methylation. These enzymes methylate a variety of substrates using S-Adenosyl-L-methionine (SAM) as the methyl group donor, leaving the common product S-adenosylhomocysteine (SAH). AptaFluor overcomes problems with current detection methods that can't detect enzymes with low turnover or high affinity for SAM. This assay is designed for high throughput screening with an easy-to-use, mix-and-read format. 

The kit comes complete with detection reagents required to measure activity. 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.

How does the AptaFluor Assay Work?

The AptaFluor Assay determines enzyme activity by directly measuring SAH produced during the methylation of a variety of substrates. By directly detecting SAH in a universal and homogenous format, it is possible to perform high throughput screening with RNA, DNA, and histone methyltransferases. 

AptaFluor technology uses a natural occurring aptamer, or riboswitch, that selectively binds with SAH. The exquisite affinity and selectivity of the riboswitch enables screening and profiling of methyltransferases with unparalleled sensitivity.

The assay is available with TR-FRET readout. 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.

Direct Detection of SAH with a TR-FRET Readout

Methyltransferase Assay Kit Schematic

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®.

Ultra-Sensitive

The most sensitive HTS methyltransferase activity assay available with an LLD of 0.6 nM SAH. This dramatically reduces enzyme usage and allows the assay to be run at or below Km for SAM.

AptaFluor Z' and SAH Titration Data and Graph

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

Run your enzyme reaction, add quenching reagent, add AptaFluor reagents, and read your plates. The assay is compatible with 96, 384, and 1536-well formats. 

AptaFluor Methyltransferase Assay Protocol Outline

Compatible with a Variety of MTase Acceptor Substrates

Assay Compatibility with a Variety of MTase Substrates

Toleration of MT acceptor substrates. 100 nM SAH-SAM standard curves were set up in the presence of nucleosomes (10 ng/μL), histone H3-3 (3 ng/μL), Histone H3 (1-21) peptide (10 μM) and Poly d (I-C) (2.5 mU/μL); control wells lacked a MT substrate.

Robust Assay Yields Quality Data

Excellent Z’ under a variety of assay conditions.

Z' for PRMT4 using Methyltransferase Assay

PRMT4 enzyme with 0.2 uM or 2 uM SAM and histone substrate as positive controls. PRMT4 and histone substrate as negative control.

Use with Physiological SAM Concentrations Lower than 100nM

SET7/9 Enzyme Titration with Linear Response Curve

Stable Detection of Methyltransferase Activity

Outstanding reagent and signal stability reduces assay variability and allows for automated workflows.

Signal Stablity of AptaFluor Assays at 3 hours, 6 hours, and Overnight

Universal – Use with Virtually Any Methyltransferase

AptaFluor Titration with multiple MTases
Methyltransferase Enzyem and Substrate Chart

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

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