TRAF6 TR-FRET Assay Kit

The TNF receptor associated factor 6 (TRAF6) TR-FRET Assay Kit is a sensitive TR-FRET Assay Kit, designed to measure TRAF6 (amino acids 51-159) auto-ubiquitination activity in a homogeneous 384 reaction format. This assay measures mono- or poly-ubiquitination. As a homogeneous assay, it requires no time-consuming washing steps and is especially suitable for high throughput screening (HTS) applications as well as real-time analyses. Read out requires a fluorescent plate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) .Figure 1.TRAF6 TR-FRET Assay Kit schematic.The assay takes advantage of biotinylated ubiquitin, which is linked to TRAF6 during auto-ubiquitination. The streptavidin-conjugated acceptor binds to ubiquitin-biotin while a Terbium-conjugated anti-GST antibody donor binds to GST-tagged TRAF6, allowing TR-FRET pairing. The resulting TR-FRET signal is directly proportional to TRAF6 mono or poly-ubiquitination.

Product Specifications

Background

Covalent conjugation of proteins 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. TRAF6 (TNF receptor-associated factor 6) is an E3 ligase, involved in inflammation and innate immunity. Inhibitors of TRAF6 activity impact the NF-κB pathway and can prove beneficial for the treatment of auto-immune and chronic inflammatory disorders, such as psoriasis and rheumatoid arthritis.

UniProt

Q9Y4K3

Format

Catalog #NameAmountStorage80301UBE1 (UBA1), Flag-Tag*50 µg-80°C80314UBCH5b, His-Tag (Human) *60 µg-80°C101597TRAF6, GST-Tag*8 µg-80°C11236Ubiquitin, His-Avi Tag, Biotin-Labeled2 x 12 µg-80°CTb-labeled donor2 x 10 µl-20°CDye-labeled acceptor2 x 10 µl-20°CATP (4 mM) 2 x 1 ml-80°CU2 Assay Buffer2 x 10 ml-80°CWhite, nonbinding, 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 TRAF6 intrachain TR-FRET Assay Kit is compatible with up to 1% final DMSO concentration.TRAF6, GST-Tag (BPS Bioscience #101597) is comprised of amino acids 51-159. Inhibitors targeting TRAF6 regions outside of the 51-159 amino acid region cannot be assessed using this assay kit.