Products/Services Used | Details | Operation |
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Gene Synthesis> | The DNA sequence for full length UCHL1C90A, UCHL1C152A, and UCHL1C90A, C152A was synthesized by <b>GenScript</b> and cloned into pET3a expression vectors. | Get A Quote |
Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In... More