DCAF15 Intrachain TR-FRET Assay Kit

The DCAF15 Intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET (Time Resolved Fluorescence Resonance Energy Transfer) Assay Kit designed to measure DCAF15 (DDB1 and CUL4-associated factor 15) auto-ubiquitination activity in a homogeneous 384-reaction format. It utilizes Europium-labeled Ubiquitin (donor) and Cy5-labeled Ubiquitin (acceptor) to complete the TR-FRET pairing. This assay measures poly-ubiquitination since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on DCAF15. As a homogenous assay, it requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time kinetics analyses.Figure 1. DCAF15 Intrachain TR-FRET Assay Kit schematicNeed us to run inhibitor screens or profile your compounds against DCAF15? Check out ourUbiquitination Screening Services.

Product Specifications

Background

Covalent conjugation to ubiquitin (Ub) is one of the major post-translational modifications that regulates 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.DDB1 and CUL4-associated factor 15 (DCAF15) is a protein that associates with CUL4A or CUL4B to form a complex with E3 ligase activity. DCAF15 interacts with and ubiquitinates the major transcription factor ZEB1, which is involved in the activation of the epithelial-mesenchymal transition (EMT) in metastatic cancer cells. Furthermore, DCAF15 is necessary for ubiquitination of the RNA splicing factor RBM39, the degradation of which leads to enhanced sensitivity of hematopoietic and lymphoid cancer cells to drug-induced cytotoxicity. Therefore, DCAF15 is an attractive potential drug target for cancer therapy. Like most E3 ligases, DCAF15 ubiquitinates itself.

UniProt

UBE1: P22314; UBCH5b: P62837; DCAF15: Q66K64

Applications

Screen molecules that inhibit DCAF15 Ubiquitin ligase activity in drug discovery HTS applicationsDetermine Inhibitor IC50Perform DCAF15 real-time kinetics

Format

Catalog #NameAmountStorage80301UBE1 (UBA1), FLAG-tag (E1) *50 µg-80°CAvoid multiple freeze/ thaw cycles80314UbcH5b, His-Tag (E2) *60 µg-80°C101497DCAF15/Rbx1/CUL4B/ DDB1/complex (E3) *130 µg-80°C78307TRF Ubiquitin Mix (200x) 50 µl-80°CATP (4 mM) 2 x 1 ml-80°CU2 Assay Buffer2 x 10 ml-80°CWhite, nonbinding Corning, low volume microtiter plateRoom Temp* 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 DCAF15 Intrachain TR-FRET Assay Kit is compatible with up to 1% final DMSO concentration. We recommend preparing the inhibitor in no higher than 5% DMSO solution in buffer and using 4 µl per well.