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. 2006 Aug 2;61(5):1187–1195. doi: 10.1111/j.1365-2958.2006.05307.x

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This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.

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Probe mechanisms of action. Probes were designed for the detection of enzymes capable of removing Ub and other Ub‐like proteins. An example of the Ub‐based probes is shown. Probes include a Ub moiety followed by either (A) a vinylmethylester or (B) a vinylmethylsulphone active group. Where needed, a haemagglutinin (HA) epitope tag is also included as an N‐terminal fusion. The catalytic groups act as electrophiles mimicking the C‐terminal end of the Ub gly motif and attracting DUB attack. DUBs become covalently and irreversibly attached to the probes through a thioether bond, thereby allowing for detection by anti‐HA antibodies in addition to a 10 kDa increase in size.