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Applications: Screen molecules that inhibit Dcaf11 Ub ligase activity in drug discovery high throughput screening (HTS) applications.Determine compound IC50 Analyze Dcaf11-driven ubiquitination.
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 E2ˆ¼Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2ˆ¼Ub to the substrate, leading to its mono- or poly-ubiquitination.DDB1 and CUL4-associated Factor 11 (Dcaf11, also known as WD Repeat Domain 23, WDR23) is a protein that associates with Cul4A or Cul4B to form a complex with E3 ligase activity. Dcaf11 E3 ligase can ubiquitinate a variety of substrates, including p21 (cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1). P21 is involved in cell cycle regulation by inhibiting CDK (cyclin-dependent kinase) and PCNA (proliferating cell nuclear antigen). It also mediates p53 response to DNA damage, and its levels have been linked to lymph node metastasis and overall survival rate to cancer. Knocking down any component of the Dcaf11 complex attenuates the ubiquitination level of p21, inhibiting osteosarcoma cell proliferation. The use of drugs targeting the Dcaf complex may be a viable cancer therapy approach.
Contraindications: The Dcaf11-driven p21 Ubiquitination Assay Kit is compatible with up to 1% final DMSO concentration. We recommend preparing the inhibitor in no higher than 4% DMSO solution in buffer and using 2.5 µl per well.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 the presence of 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. MEM, which lacks these components, does not affect AlphaScreen® assays.
Description: The Dcaf11-driven p21 ubiquitination assay kit is a sensitive AlphaLisa® high-throughput screening (HTS) assay kit, designed to measure Dcaf11 (DDB1- and Cul4-associated factor 11) E3 ligase activity in a homogeneous 384 reaction format. The assay kit comes with enough biotinylated ubiquitin, ATP, GST-tagged p21, assay buffer, detection buffer, purified UBE1 (E1), UbcH5b (E2), and Rbx1/Cul4B/Dcaf11/Ddb1 complex (E3) for 384 reactions. The assay detects mono-ubiquitination and poly-ubiquitination of p21.First, E1 and E2 enzymes are incubated with the E3 complex and with the target protein p21 (GST-tag) in the presence of biotin-conjugated ubiquitin and ATP. Ubiquitination of p21 occurs in a multistep ubiquitin transfer from E1 to E2 to E3, and E3-mediated conjugation of ubiquitin to p21. Next, glutathione-acceptor beads are added, followed by streptavidin-conjugated donor beads, and Alpha-counts are measured. The increase in Alpha-counts is proportional to the mono- or poly-ubiquitination of the GST-tagged p21.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Uniprot: UBE1: P22314; UbcH5b: P62837; Rbx1: P62877; Cul4B: Q13620; Dcaf11: Q8TEB1; Ddb1: Q16531
Warnings: Avoid freeze/thaw cycles
Biosafety Level: Not applicable (BSL-1)
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