In accordance with previous results [28] , we observed a non-linear decrease in electrophoretic mobility of the ISG15VS adducts. The ISG15 probe has a mass of 17.4 kDa, whereas the size increase observed for each of the DUBs investigated here is in the order of 25-110 kDa, when bound to ISG15VS. The correlation between the abnormal shift and the initial mass of the unmodified DUB suggests that the decrease in gel mobility is based on steric properties of the branched adduct, and is not caused by covalent modification of a single DUB by multiple ISG15VS molecules. In fact, in our sample set, the observed size increase of the ISG15VS-DUB adducts based on SDS-PAGE very closely matched a logarithmic equation ( Figure 3A and see Methods). To further verify that DUBs are modified by only a single ISG15VS probe per molecule, we replaced the catalytic cysteine at position 114 in USP14 with a serine residue. As expected, this mutation abolished all labeling ( Figure 3B ). Our assay was conducted in IVT lysate and the size increase of the ISG15VS adduct could potentially reflect modification of USP14 by additional factors. However, even USP14 that was recombinantly expressed in bacteria and .95% pure showed the same abnormal electrophoretic mobility for its ISG15VS adduct ( Figure 3C ). Mass spectrometry confirmed the monovalent modification of purified USP14 by ISG15VS and excluded covalent binding of additional factors to the complex ( Figure 3D ). Collectively, these experiments establish that the observed shift in apparent molecular mass of the ISG15VS-DUB adducts is solely a consequence of its unusual electrophoretic behavior. It also underscores the uncertainties in estimating the degree of modification of a target protein with UbLs by SDS-PAGE alone.