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Sensitive Detection of PRMT4 with the AptaFluor® Methyltransferase Assay

PRMT4 Assay - An AptaFluor SAH Assay Application

The AptaFluor Methyltransferase Assay measures PRMT4 (also known as CARM1 or protein arginine N-methyltransferase 4) activity via direct detection of SAH produced during methylation. The AptaFluor Assay uses a naturally occurring SAH-sensing riboswitch that binds SAH with nanomolar affinity and exquisite selectivity.

The AptaFluor Assay accommodates SAM concentrations as low as 100 nM and as high as 5 μM. The greater sensitivity of the assay allows practical detection using significantly less PRMT4. 

The kit comes complete with the detection reagents required to measure activity. PRMT4 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 This PRMT4 Assay Work?

The PRMT4 Assay determines PRMT4 activity by directly measuring SAH produced during the methylation of 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.

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. 

The PRMT4 assay is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS. It is available with a TR-FRET readout.

Direct Detection of SAH to Measure PRMT4 Enzymatic Activity

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

PRMT4 Assay Schematic

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 PRMT4 assay is compatible with 96, 384, and 1536-well formats.

AptaFluor Methyltransferase Assay Protocol Outline

Applications

  • Measure enzymatic activity of PRMT4
  • Screen Compound Libraries for PRMT4 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 = 0.82 using 0.2 μM SAM & 0.83 using 2 μM SAM.

Use with Varying SAM Concentrations

Detection of PRMT4 initial velocity at 200 nM SAM and 2 μM SAM. The assay demonstrates linearity when raw data is converted to SAH using a standard curve. Assays were run in LV 384-well white plates and read on a PheraStar Plus. The complete assay had a 20 μL volume after addition of detection reagents. The PRMT4 enzyme reactions contained 10 µM H3.3 peptide in addition to PRMT4 and took place for 120 minutes at 30°C.  

PRMT4 Enzyme Titration @ 200 nM SAM

Linear Response

Linear Response Curve PRMT4 at 200 nM

PRMT4 Enzyme Titration @ 2 μM SAM

PRMT4 Assay Enzyme Titration Data at 2 μM

Linear Response

Linear Response Curve PRMT4 Assay at 2 uM Concentration

PRMT4 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 PRMT4 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 PRMT4 Activity Assays

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

The Role of PRMT4 As a Therapeutic Target

The protein arginine methyltransferase (PRMT) family catalyzes protein arginine methylation. Many cancers link to dysregulation of protein arginine methylation, making PRMTs an important target for anti-cancer treatments. PRMT4 is a type I PRMT that catalyzes asymmetric dimethylarginine and monomethylarginine. It is a coactivator for several nuclear receptors and transcription factors. (Du et al, 2021)

PRMT4 is important for many cellular processes, including transcription activation, RNA processing, tumorigenesis & cancer progression, cell growth/differentiation, and apoptosis. Breast cancer, lung cancer, colorectal cancer, and prostate cancer have all linked to PRMT4 mutations. (Du et al, 2021)

The AptaFluor PRMT4 Assay is a powerful tool to screen compound libraries for PRMT4 modulators to help find new therapies for disease. 

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