Ubi-Trans™ UBA6-UbcH5b TR-FRET Assay Kit

Ubiquitination is a multistep process that involves E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes) and E3 (ubiquitin ligase) enzymes. In the first step, an E1-ubiquitin thioester bond is formed between the C-terminal glycine carboxyl group of ubiquitin and the cysteine in the active site of the E1 enzyme in an ATP-dependent reaction. Next, the E1 enzyme transfers the activated ubiquitin to the cysteine residue of the E2 enzyme to form an E2-ubiquitin thioester-linked intermediate by transesterification. Finally, the E2 enzyme transfers ubiquitin to the substrate protein with the help of an E3 ligase. UBA6 (ubiquitin-like modifier activating enzyme 6) is an E1 enzyme that activates ubiquitin but also has the unique feature of being able to act on FAT10 (also known as ubiquitin D or human leukocyte antigen-F adjacent transcript 10) . UBA6 is present in most tissues at very low levels, however it is upregulated during dendritic cell maturation and hyperthermic stress. UBA6 can transfer ubiquitin to UbcH5b (also known as Ubiquitin-conjugating Enzyme E2 D2, UBE2D2) . UbcH5b is overexpressed in certain cancer types. It ubiquitinates the tumor-suppressor proteins p53 and p62, promoting their degradation. The UbcH5b-p62 complex confers therapeutic resistance in triple negative breast cancer cells. UbcH5b is also involved in endocytosis-mediated degradation of MHC (major histocompatibility complex) class I molecules, and overexpression has been found in inflammatory bowel disease. Inhibitors of ubiquitination of UbcH5b by UBE6 may prove beneficial in cancer and auto-immune disease treatment.

Catalog #NameAmountStorage80303UBA6 (UBE1L2), FLAG-Tag*4.8 µg-80°C80314UbcH5b, His-Tag (Human) *5 µg-80°CBiotin-Ubiquitin80 µl-80°C4 mM ATP1 ml-80°C78856U2 Assay Buffer2 x 10 ml-80°C30017Anti-His Tb-Labeled Donor10 µl-20°CDye-Labeled Acceptor10 µl-20°C79969White, nonbinding, low volume microtiter plateRoom Temp* The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.