K63 diUb PA

Potent, irreversible and specific inhibitor based on K63 linked diUb that targets the S2 pocket of deubiquitinating enzymes (DUBs). The C-terminus of the proximal Ub is equiped with the propargylamide (PA) electrophile and the isopeptide bond between the two Ub proteins is replaced by a DUB resistant mimic.

Important: sample preparation.
§ dissolve the powder in as little DMSO as possible (e.g. 20 mg/mL) and add this DMSO stock slowly to milliQ (please note the order of addition).
§ buffer with e.g. 1M HEPES to 50 mM HEPES. In general HEPES and Tris buffers are standard for DUB assays.
§ add NaCl if required (e.g. from a 5M stock). Please note that certain DUBs react different to low or high NaCl concentrations.
§ 2 - 5 mM DTT can be used as reducing agent for the DUB. § a final buffered stock of for example 0.5 mg/mL contains 2.5 vol% DMSO; in general DMSO concentrations of up to 5 vol% are well tolerated by DUBs.
§ if required, total removal of DMSO is accomplished by dialysis or spin-filtration (3 kDa cut-off membrane).