Sensitive Detection of PRMT4 with the AptaFluor® Methyltransferase Assay

PRMT4 Assay Methods

Ordering Information

Assay Services

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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. Transcreener 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)

Learn More About The AptaFluor Assay

AptaFluor SAH TR-FRET Assay Tech Manual

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 K_M for SAM.

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

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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

PRMT4 Enzyme Titration @ 2 μM SAM

PRMT4 Assay Enzyme Titration Data at 2 μM

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Linear Response

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.

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 k_off 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.

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

Supplier	Instrument	TR-FRET Assays
	TriStar²S LB 942	Validated
	Tristar 5	In Review
	Mithras² LB 943	Validated

	Cytation^TM 5	Validated
	Cytation^TM 3	Validated
	Cytation^TM 1	Validated
	Synergy^TM H1	Validated
	Synergy^TM 2/H4/4	Validated
	Synergy^TM HTX	Not Capable
	Synergy^TM Neo 2	Validated

	POLARstar^® Omega	Validated
	FLUOstar^® Omega	Validated
	PHERAstar^® FSX	Validated
	PHERAstar^® Plus/FS	Validated
	CLARIOstar^® /Plus	Validated
	VANTAstar^TM	Validated

	Sense	In Review

	Analyst^® GT/HT	Validated
	Gemini^® XPS/EM	Not Capable
	SpectraMax^® M2/M2e	Not Capable
	SpectraMax^® M5/M5e/FlexStation^® 3	Validated
	SpectraMax^® Paradigm	Contact Us
	SpectraMax^® iD3/iD5	Validated
	SpectraMax^® i3x	In Review

	EnVision^®/EnVision^® Xcite	Validated

	Infinite^® M1000/M1000Pro/Safire2^TM	Validated
	Infinite^® M200	Not Capable
	Infinite^® F500	Validated
	Infinite^® F200/Ultra^evolution	Contact Us
	Spark^TM 10M	Validated

What's Included


Component	Notes
SAM	SAM supplied in the kit should be used for MT enzyme reactions and to create a SAM/SAH standard curve.
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.

What You Will Need


Component	Notes
PRMT4 Enzyme	We have successfully used recombinant PRMT4 methyltransferase enzyme from Reaction Biology #HMT-11-120.
Substrate	We successfully used recombinant Histone H3.3 from Reaction Biology #HMT-11-134. The PRMT4 enzyme reaction contained 10 μM H3.3 peptide.
Enzyme Buffer	20 mM Tris (pH 8.5), 5 mM MgCl₂, 1 mM DTT, and 100 mM NaCl. 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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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.

Cat. No.	Product Description	Price	Add to Cart
3023-B	AptaFluor SAH Methyltransferase Assay (100 Assay, 96 Well)	$399.00	Add to Cart
3023-1K	AptaFluor SAH Methyltransferase Assay (1,000 Assay, 384 Well)	$1,299.00	Add to Cart
3023-10K	AptaFluor SAH Methyltransferase Assay (10,000 Assay, 384 Well)	$7,399.00	Add to Cart

Assay Plates for TR-FRET

Cat. No.	Product Description	Price	Add to Cart
2234	Corning 96-Well White Assay Plates (#3642), 3-Pack (200+ Assays)	$55.00	Add to Cart
2231	Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)	$65.00	Add to Cart
2232	Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)	$529.00	Add to Cart

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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.

PRMT4 Assay – AptaFluor Application

Sensitive Detection of PRMT4 with the AptaFluor® Methyltransferase Assay

PRMT4 Assay Methods

Ordering Information

Assay Services

Related Resources

PRMT4 Assay - An AptaFluor SAH Assay Application

How Does This PRMT4 Assay Work?

Direct Detection of SAH to Measure PRMT4 Enzymatic Activity

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

The Most Sensitive HTS Methyltransferase Assay

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

Applications

Features

Robust Assay Yields Quality Data

Use with Varying SAM Concentrations

PRMT4 Enzyme Titration @ 200 nM SAM

Linear Response

PRMT4 Enzyme Titration @ 2 μM SAM

Linear Response

PRMT4 Assay Services

Lead Discovery Services Include:

Contact Us to Learn More

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

What's Included

What You Will Need

AptaFluor SAH Methyltransferase Assay Kits - For PRMT4 Activity Assays

Assay Plates for TR-FRET

The Role of PRMT4 As a Therapeutic Target

Related Resources