Description
Product Usage Information
trFluor™ Europium Cryptate conjugates are ideal for protein-protein interaction assays, pair-based immunoassay technologies, or high-throughput screening where greater sensitivity and quantitative detection is needed. The long fluorescent decay time of Europium Cryptate results in improved signal to background over fluorescent dye conjugates. Methods utilizing Europium Cryptate conjugates include, but are not limited to, TR-FRET, where Europium Cryptate-labeled donors are paired with red fluorophore-labeled acceptor antibodies, such as Alexa Fluor ® 647 or APC. Please contact us if you require an acceptor antibody clone conjugated to a red dye at a custom concentration, a carrier-free formulation, or a more customized packaging solution. Note: Addition of potassium fluoride (KF) in the assay dilution buffer range of 40-400 mM is recommended for enhanced fluorescent signal. Optimal dilutions and working concentrations of both the conjugated antibodies and the KF should be determined by the end user.
Storage
Supplied in PBS (pH 7.2), less than 0.1% sodium azide, and 2 mg/mL BSA. Store at 4°C. Do not aliquot. Protect from light. Do not freeze.
Specificity / Sensitivity
TUBE-RAD23A binds to endogenous levels of proteins containing polyubiquitinated chains. It binds to K48- and K63-branched tetraubiquitin chains.
Source / Purification
TUBE, designed from the ubiquitin binding domain of RAD23A, was produced in . Pan-branch Ubiquitin TUBE-RAD23A protein also contains a GST-tag, His-tag, and V5-tag.
Background
Ubiquitin is a conserved polypeptide unit that plays an important role in the ubiquitin-proteasome pathway. Ubiquitin can be covalently linked to many cellular proteins by the ubiquitination process, which targets proteins for degradation by the 26S proteasome. Three components are involved in the target protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thiolester complex with the activation component E1; the activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH of the target protein lysine residue (1-3). The ubiquitin-proteasome pathway has been implicated in a wide range of normal biological processes and in disease-related abnormalities. Several proteins such as IκB, p53, cdc25A, and Bcl-2 have been shown to be targets for the ubiquitin-proteasome process as part of regulation of cell cycle progression, differentiation, cell stress response, and apoptosis (4-7). Substrate proteins are linked to ubiquitin using seven distinct ubiquitin lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63). Formation of a polyubiquitin chain occurs when a lysine residue of ubiquitin is linked to the carboxy-terminal glycine of another ubiquitin. Proteins polyubiquitinated at specific lysine residues display a tendency to be targeted for different processes; K48-linked polyubiquitin chains mainly target proteins for proteasomal degradation, while K63-linked polyubiquitin chains regulate protein function, subcellular localization, or protein-protein interactions (8). K63-linked polyubiquitin chains exert nonproteolytic functions , such as protein trafficking, kinase/phosphatase activation, and DNA damage control, all of which might be important in regulation of cancer survival and development (9,10). Ubiquitin-associated (UBA) domains are protein regions that interact with ubiquitin. Tandem-repeated ubiquitin-binding entities (TUBEs) were designed by using four tandem UBA domains, based on the theory that tetraubiquitin chains are a minimum requirement for efficient proteasomal degradation (11). TUBEs designed with UBA domains from UBQLN1 and RAD23A bind to K48- and K63-linked tetraubiquitin chains and can be used to efficiently purify ubiquitylated proteins from cell extracts (12).
