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Tetra-ubiquitin (K63-linked)
Polyubiquitin chains, linked through specific lysine residues, are useful tools for investigating, amongst other things, the specificity and reactivity of deubiquitinylating enzymes (DUBs) and the recognition and interaction of polyubiquitin modified proteins with ubiquitin binding domain (UBDs) containing proteins. K63-linked tetra-ubiquitin (Ub4) is assembled from two di-ubiquitin (Ub2) units. One Ub2 is made by incubating E1 and Ubc13-Mms2 with equal concentrations of ubiquitin capped at the future proximal chain terminus (Asp77-ubiquitin) and ubiquitin capped at the future distal chain terminus (K48R/K63R-ubiquitin). A second Ub2 is made similarly, but with Asp77-ubiquitin and ubiquitin blocked at the future distal chain terminus with a C63 residue (K63C-ubiquitin). The proximal or distal terminus of the resulting dimers are deblocked by treatment with yeast ubiquitin hydrolase-1 (YUH-1) or ethyleneimine, respectively. In the second cycle of synthesis, tetra-ubiquitin (Ub4) is made by incubating the two singly-deblocked dimers with E1 and Ubc13- Mms2. Following purification by anion and cation exchange chromatography, the proximal terminal Asp77 is removed by treatment with YUH-1.
Product Specification
Alternative Name: Ub4
Source: Produced in E. coli.
Concentration: 2.5mg/ml
Formulation: Liquid. In 50mM TRIS, pH 7.6, containing 1mM EDTA.
Short Term Storage: -20°C
Long Term Storage: -20°C
Use/Stability: Stable for at least 1 year after receipt when stored at -80°C. It is recommended that the product be reconstituted at a moderately high protein concentration (>1mg/mL) and in the presence of buffer. If a more dilute reagent is required, the inclusion of an inert carrier protein (e.g. ovalbumin at 0.5-1.0mg/mL) in the storage buffer is recommended in order to minimise loss of the reagent due to absorption.
Handling: Avoid freeze/thaw cycles. After opening, prepare aliquots and store at -80°C. |