Protocol for integrated ubiquitination analysis of in vitro E3 ligase-DUB regulation and in vivo ubiquitin chain linkage characterization

Here, we present a protocol for the recombinant production of DTX3L and USP28, along with in vitro and in vivo procedures for examining their regulatory functions. We describe steps for reconstituting DTX3L-dependent ubiquitination in vitro with purified UBA1, UbE2D1, ubiquitin, and DTX3L and assessing USP28-mediated deubiquitination. We then detail procedures for the in vivo detection of ubiquitin chain linkage-type through co-immunoprecipitation and Western blotting. This adaptable approach enables analysis of regulatory crosstalk between E3 ligases and deubiquitinases within the ubiquitin system. For complete details on the use and execution of this protocol, please refer to Mennerich et al. 1