In vitro ubiquitination: self-ubiquitination, chain formation, and substrate ubiquitination assays

Ubiquitination of proteins in vitro has evolved as an indispensable tool for the functional analysis of this posttranslational modifi cation. In vitro ubiquitination is particularly helpful to study conjugation mechanisms. The effi ciency of the ubiquitination reaction depends in part on the quality of the enzymes utilized. Here we introduce the assay developed in our lab to study HECT E3 ligases. It involves bacterially expressed E1, His-tagged Ube2D3 (also called UbcH5c, the best E2 for Nedd4), untagged Nedd4, and untagged ubiquitin (Ub). As tags may impair specifi c activity of the enzymes or even interfere with the enzymatic reaction, they should be avoided, removed, or kept to a minimal size whenever possible, unless proven to be without consequence. The protocol described here is suitable for other E3 ligases capable of forming Ub chains as pseudo-product of the enzyme reaction. It is also adapted to include substrates. In this case, substrates should be tagged and purifi ed after the reaction is completed to allow the detection of the ubiquitinated products.