PRMT5 Homogeneous Assay Kit

Catalog #

52052

$1,865 *

Size: 384 reactions

Qty

Bulk/Custom

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*US Pricing only. For international pricing, please contact your local distributor.

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Description

The PRMT5 Homogeneous Assay Kit is designed to measure PRMT5 activity for screening and profiling applications. PRMT5 is a histone methyltransferase that methylates histone H4-R3. The Assay Kit relies on a highly specific antibody that recognizes the methylated form of the substrate. With this kit, only three simple steps on a microtiter plate are required for methyltransferase detection. First, a sample containing PRMT5 enzyme is incubated with the biotinylated substrate for two hours. Next, acceptor beads and primary antibody are added, then donor beads, followed by reading the Alpha-counts.

Illustration of the assay principle: The PRMT5/MEP50 enzyme methylates the biotinylated histone H4 peptide substrate. The methylated form of the peptide is recognized by a highly specific rabbit antibody, which binds to anti-Rabbit acceptor beads, while the biotin binds to streptavidin-conjugated donor beads, bringing the acceptor and donor beads in close proximity. Upon excitation of the donor bead a singlet oxygen is generated by the donor bead, which excites the acceptor bead and emits light proportionally to the level of interaction. AlphaLISA™ immunoassays are a no-wash alternative to ELISA immunoassays. These assays are robust and ideal for a minimal hands-on approach.

*NOTE: As of April 2023, this kit has been re-optimized for performance.

This product has been cited 1 time.

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Synonyms

PRMT5, HRMT1L5, IBP72, JBP1, SKB1, PRMT-5

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Product Data Gallery

PRMT5 Activity

Product Info

Storage and Usage

Citations1

Assay Kit Format

AlphaLISA®

Supplied As

The PRMT5 Homogeneous Assay Kit comes in a convenient AlphaLISA® format, with biotinylated histone H4 peptide substrate, primary antibody, methylation assay buffer, and purified PRMT5/MEP50 complex for 384 enzyme reactions.

Materials Required But Not Supplied

AlphaLISA® anti-rabbit IgG acceptor beads, 5 mg/ml (PerkinElmer #AL104C)
AlphaScreen® Streptavidin-conjugated donor beads, 5 mg/ml (PerkinElmer #6760002)
Optiplate-384 (PerkinElmer #6007290)
Dithiothreitol, DTT (0.5M)

Format

Catalog #	Component	Amount	Storage
51045	PRMT5/MEP50 FLAG-Tag, His-Tag (HEK293-derived)*	10 μg	-80°C	Avoid multiple freeze/ thaw cycles!
52120-A	S-adenosylmethionine (20 µM)	2 x 250 μl	-80°C
52150-3	Primary Antibody 4-3**	100 μl	-80°C
	Biotinylated histone H4 peptide substrate	500 reactions	-80°C
	PRMT5 Assay buffer (4x); add DTT before use	3 ml	-20°C
52301	Detection Buffer (4x)	2 ml	-20°C

*The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.

** The detection antibody for this kit was changed in January 2017.

UniProt #

O14744

Background

Protein arginine N-methyltransferase 5 (PRMT5, also known as ANM5) is part of the methylosome complex, composed of MEP50, PRMT5, WDR77 and CLNS1A. This complex methylates/demethylates specific arginine residues in histones H2AR3, H4R3, H3R2, and H3R8. It also methylates various proteins such as SUPT5H (Transcription elongation factor SPT5), Piwi proteins, RPS10, and more. MEP50 protein is the non-catalytic component of the complex and is a core component of the complex that mediates interactions with binding partners and substrates.

References

Stopa, N., et al. 2015. Cell Mol Life Sci. 72(11):2041-59.

Storage/Stability

This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.

Applications

Screen molecules in high-throughput applications
Determine compound IC₅₀
Perform real-time kinetic analyses

Shipping Temperature

-80°C

Notes

Troubleshooting Guide
Visit bpsbioscience.com/assay-kits-faq for detailed troubleshooting instructions. For all further questions, please email [email protected]

Contraindications

Green and blue dyes that absorb light in the AlphaScreen® signal emission range (520-620 nm), such as Trypan Blue, interfere with the assay. Avoid using potent singlet oxygen quenchers such as sodium azide (NaN3) or metal ions (Fe2+, Fe3+, Cu2+, Zn2+ and Ni2+). The presence of >1% RPMI 1640 culture medium leads to a signal reduction due to the presence of excess biotin and iron in this medium. MEM, which lacks these components, does not affect AlphaScreen® assays.

Warnings

Avoid freeze/thaw cycles.

Disclaimers and Limitations

BPS Bioscience assay kits are validated using the listed components according to our specific protocol. Any deviations to kit components or protocols, such as using different proteins, cell/tissue lysates, or buffers (including using the same component from other commercial sources) will void any warranties on the performance of the assay kit and is not recommended.

End-users should immediately report any issues to [email protected] upon receipt of the kit, such as missing components, damaged vials, no dry ice, etc.

Scientific Category

Methyltransferase

Regulatory Status

Research Use Only