MDM2-Driven p53 Ubiquitination Assay Kit

Catalog #
82179
$1,750 *
Size: 384 reactions
Qty
*US Pricing only. For international pricing, please contact your local distributor.
Purchase
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.

Product Info
Storage and Usage
Citations
Assay Kit Format
AlphaLISA®
Species
Human
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.