MDM2-Driven p53 Ubiquitination Assay Kit

Catalog #

82179

$1,750 *

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 MDM2-Driven p53 Ubiquitination Assay kit is a sensitive AlphaLisa® high-throughput screening (HTS) assay designed to measure MDM2 (mouse double minute 2 homolog) E3 ligase activity in a homogeneous 384 reaction format. The assay kit comes with enough biotinylated ubiquitin, ATP, FLAG-tagged p53, assay buffer, detection buffer, purified UBE1 (E1), UbcH5b (E2), and MDM2 (E3) for 384 reactions. The assay can detect mono-ubiquitination and poly-ubiquitination of p53.

Figure 1: MDM2-Driven p53 Ubiquitination Assay Kit schematic.
E1 and E2 enzymes are incubated with the E3 complex and FLAG-tagged p53, in the presence of biotin-conjugated ubiquitin and ATP. Ubiquitination of p53 occurs in a multistep ubiquitin transfer from E1 to E2 to E3, and E3-mediated conjugation of ubiquitin to p53. Next, acceptor beads are added, followed by streptavidin-conjugated donor beads. Alpha-counts are then measured. The increase in Alpha-counts is proportional to the mono- or poly-ubiquitination of the FLAG-tagged p53.

Need us to run inhibitor screens or profile your compounds against MDM2-Driven p53? Check out our Ubiquitination Screening Services.

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Product Data Gallery

MDM2-driven p53 ubiquitination.

Product Info

Storage and Usage

Citations

Assay Kit Format

AlphaLISA®

Materials Required But Not Supplied

AlphaLISA® anti-FLAG acceptor beads (Perkin Elmer #AL112C)
AlphaScreen® Streptavidin-conjugated donor beads (Perkin Elmer #6760002S)
Optiplate-384 (Perkin Elmer #6007290)
AlphaScreen® microplate reader
DNAse free water
Orbital Shaker

Format

Catalog #	Name	Amount	Storage
100402	UBE1 (UBA1), GST-Tag*	25 µg	-80°C
80314	UBCH5b, His-Tag (Human)*	50 µg	-80°C
100409	MDM2, GST-Tag*	1 µg	-80°C
100412	p53, FLAG-Tag*	2 x 2 µg	-80°C
	Biotin-Ubiquitin	400 µl	-80°C
	10 mM ATP	400 µl	-80°C
	U2 Assay Buffer	2 x 10 ml	-80°C
	4x U2 Detection Buffer	2 x 2 ml	-20°C

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

Background

Covalent conjugation to ubiquitin (Ub) is one of the major post-translational modifications regulating protein stability, function, and localization. Ubiquitination is the concerted action of three enzymes: a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The specificity and efficiency of ubiquitination are largely determined by the E3 enzyme, which directs the last step of the Ub-conjugating cascade by binding to both an E2∼Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2∼Ub to the substrate, leading to its mono- or poly-ubiquitination.

The p53 tumor suppressor protein is regulated by its interaction with MDM2, which serves as a ubiquitin ligase (E3) to target p53 for degradation. MDM2 ubiquitinates p53, resulting in the rapid degradation of p53 through the Ub–proteasome pathway. MDM2-mediated destabilization and inactivation of p53 are thought to play a critical role in several human cancers. The disruption of the MDM2-p53 interaction has been regarded as an attractive strategy for anticancer drug discovery.

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 that inhibit MDM2 Ubiquitin ligase activity in drug discovery high-throughput screening (HTS) applications,
Determine compound IC₅₀.
Perform MDM2-driven ubiquitination studies.

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

The MDM2-Driven p53 Ubiquitination Assay Kit is compatible with up to 1% final DMSO concentration.
Green and blue dyes that absorb light in the AlphaScreen® signal emission range (λ=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 the culture medium RPMI 1640 at >1% leads to signal reduction due to the presence of excess biotin and iron in this medium. Media such as 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

Cancer Therapy Target

Regulatory Status

Research Use Only