ChoosE2-Opti™ Intrachain TR-FRET Assay Kit

ChoosE2-Opti™ Intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit, designed to identify E2 enzyme (s) that are functional partners for apurifiedE3 ubiquitin ligase of interest and lead to its self-polyubiquitination. The assay kit contains five E2 enzymes to be tested with the E3 ligase of interest, and two E3 ligases (SMURF1 and MDM2) to use as internal controls. It also contains an E1 enzyme, ATP, an optimized TRF Ubiquitin Mix, and a universal buffer.It utilizes a Europium-labeled Ubiquitin Donor as well as Cy5-labeled Ubiquitin Acceptor to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into polyubiquitin chains, this FRET-based assay requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time kinetics analyses.Figure 1: ChoosE2-Opti intrachain TR-FRET Assay Kit schematicNeed us to run inhibitor screens or profile your compounds against ChoosE2-Opti™? Check out ourUbiquitination Screening ServicesorDNA Replication & Repair 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 E2-E3 complex where the E3 enzyme directs the last step of the Ub-conjugating cascade by binding to both an E2∼Ub conjugate and a substrate protein.

UniProt

UBE1: P22314, UbcH5a: P51668, UbcHb: P62837, UbcH5c: P61077, UbcH6: P51965, UbcH7: P68036, MDM2: Q00987, SMURF1: Q9HCE7

Applications

Identify E2 enzymes that are partners of an E3 ligase of interestAssess the activity of new E2-E3 protein pairsScreen new E3 ligases and variants in HTS applicationsScreen molecules that inhibit/activate E2 enzyme activity in HTS applications, Determine compound IC50Analyze E3 ligase activity in real-time

Format

Catalog #NameAmountStorage80301UBE1 (UBA1), FLAG-Tag*50 µg-80°CAvoid multiple freeze/ thaw cycles80315UbcH5a (UBE2D1), His-Tag*15 µg-80°C80314UbcH5b, His-Tag*15 µg-80°C80313UbcH5c, His-Tag*15 µg-80°C80316UbcH6 (UBE2E1), His-Tag*20 µg-80°C80318UbcH7, His-Tag (Sf9 derived) *15 µg-80°C100409MDM2, GST-Tag*5 µg-80°C80402SMURF1, FLAG-Tag*5 µg-80°C78307TRF Ubiquitin Mix (200x) 50 µl-80°CATP (4 mM) 2 x 1 ml-80°C78269U2 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.The Ubiquitin Mix is sourced from South Bay Bio LLC.

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

ChoosE2-Opti™ Intrachain TR-FRET Assay Kit is not suitable for measuring mono-ubiquitination. Weak signals may be obtained for multi-mono-ubiquitination or for short poly-ubiquitin chains.This kit is compatible with up to 1% final DMSO concentration. We recommend preparing the inhibitor in no higher than 5% DMSO solution in assay buffer and using 4 µl per well.The E3 ligase used in the assay should be purified. The assay will not perform well when using cell lysates due to the presence of other E3 ligases and ubiquitin.