JARID1A (KDM5A) Homogeneous Assay Kit

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Catalog #

50510

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Description

The JARID1A Homogeneous Assay Kit is designed to measure JARID1A activity for screening and profiling applications. JARID1A, also known as RBBP2 and KDM5A, is a JumonjiC (JmjC) and ARID domain-containing histone lysine demethylase that exhibits demethylation activity toward H3-K₄Me² and H3-K₄Me³. The JARID1A Homogeneous Assay Kit comes in a convenient AlphaLISA^® format (Scheme 1), with biotinylated histone H3 peptide substrate, primary antibody, demethylase assay buffer, and purified JARID1A for 96 or 384 enzyme reactions. The key to the JARID1A Homogeneous Assay Kit is a highly specific antibody that recognizes demethylated substrate. With this kit, only three simple steps on a microtiter plate are required for methyltransferase detection. First, a sample containing JARID1A enzyme is incubated with the biotinylated substrate. Next, acceptor beads and primary antibody are added, then donor beads, followed by reading the Alpha-counts.

Scheme 1: Our histone demethylase assays utilize highly specific antibodies that recognize demethylated histone substrate. First, a sample containing the enzyme is incubated with a biotinylated substrate. Next, acceptor beads and primary antibody are added, then donor beads, followed by reading the Alpha-counts, as shown below.

Scheme 1: Our histone demethylase assays utilize highly specific antibodies that recognize demethylated histone substrate. First, a sample containing the enzyme is incubated with a biotinylated substrate. Next, acceptor beads and primary antibody are added, then donor beads, followed by reading the Alpha-counts, as shown above.

Need us to run inhibitor screens or profile your compounds against JARID1A (KDM5A)? Check out our Demethylase Screening Services.

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Synonyms

JARID1A, RBBP2, KDM5A

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Product Data Gallery

Product Info

Storage and Usage

Citations

Assay Kit Format

AlphaLISA®

Supplied As

The JARID1A Homogeneous Assay Kit comes in a convenient AlphaLISA^® format, with biotinylated histone H3 peptide substrate, primary antibody, demethylase assay buffer, and purified JARID1A for 96 or 384 enzyme reactions.

Materials Required But Not Supplied

AlphaLISA anti-rIgG acceptor beads, 5 mg/ml (PerkinElmer #AL104C)
AlphaScreen Streptavidin-conjugated donor beads, 5 mg/ml (PerkinElmer #6760002S)
Optiplate -96 (PerkinElmer #6005290) or Optiplate -384 (PerkinElmer #6007290)
AlphaScreen microplate reader
Adjustable micropipettor and sterile tips

Format

96 reactions:

Catalog #	Component	Amount	Storage
50110	JARID1A (KDM5A)	2 x 10 µg	-80°C	Avoid freeze/ thaw cycles
52140M4	Primary antibody 13-4	5 µl	-80°C
	Biotinylated histone H3 peptide substrate (JARID)	300 rxns	-80°C
52407	4x HDM Assay Buffer 2	3 x 1 ml	-80°C
	4x Detection buffer D	2 ml	-20°C

384 reactions:

Catalog #	Component	Amount	Storage
50110	JARID1A (KDM5A) *	60 µg	-80°C	Avoid freeze/ thaw cycles
52140M4	Primary antibody 13-4	20 µl	-80°C
79840	Biotinylated histone H3 peptide substrate	500 rxns	-80°C
52407	4x HDM Assay Buffer 2	3 ml	-80°C
	4x Detection buffer D	2 ml	-20°C

*The concentration of JARID1A is lot-specific and will be indicated on the tube containing the enzyme

UniProt #

P29375

Background

JARID1A, also known as RBBP2 and KDM5A, is a JumonjiC (JmjC) and ARID domain-containing histone lysine demethylase that exhibits demethylation activity toward di- and trimethyl-lysine 4 (H3K4me2/3) on histone H3.

References

1. DiTacchio L, Le HD, Vollmers C et al. Science 2011; 333(6051):1881-5.

Storage/Stability

At least 6 months from date of receipt when stored as directed.

Instructions for Use

See assay kit data sheet for detailed protocol.

Applications

Useful for studying enzyme kinetics and screening small molecular inhibitors for drug discovery and HTS applications.

Shipping Temperature

-80°C

Notes

AlphaScreen^® and AlphaLISA^® are registered trademarks of PerkinElmer, Inc.

Contraindications

Avoid green and blue dyes that absorb light in the Alphascreen^® signal emission range (520-620 nm), such as Trypan Blue. Avoid the use of the 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

Demethylase

Regulatory Status

Research Use Only