MDM2-Driven p53 Ubiquitination Assay Kit

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 ourUbiquitination Screening Services.

Product Specifications

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.

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.

Format

Catalog #NameAmountStorage100402UBE1 (UBA1), GST-Tag*25 µg-80°C80314UBCH5b, His-Tag (Human) *50 µg-80°C100409MDM2, GST-Tag*1 µg-80°C100412p53, FLAG-Tag*2 x 2 µg-80°CBiotin-Ubiquitin400 µl-80°C10 mM ATP400 µl-80°CU2 Assay Buffer2 x 10 ml-80°C4x U2 Detection Buffer2 x 2 ml-20°C*The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.

Shipping Conditions

-80°C

Storage Conditions

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

Notes

Troubleshooting GuideVisitbpsbioscience.com/assay-kits-faqfor 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.