Diagnosing and mitigating method-based avidity artifacts that confound polyubiquitin-binding assays.

Polyubiquitination is a complex form of posttranslational modification responsible for the control of numerous cellular processes. Many ubiquitin-binding proteins recognize distinct polyubiquitin chain types, and these associations help drive ubiquitin-signaling pathways. There is considerable interest in understanding the specificity of ubiquitin-binding proteins; however, because of the multivalent nature of polyubiquitin, affinity measurements of these interactions that rely on affixing ubiquitin-binding proteins to a surface can display artifactual, method-dependent avidity, or "bridging." This artifact, which is distinct from biologically relevant, avid interactions with polyubiquitin, is commonplace in such polyubiquitin-binding measurements and can lead to dramatic overestimations of binding affinities for particular chain types, and thus, incorrect conclusions about specificity. Here, we use surface-based measurements of ubiquitin binding in three model systems to illustrate bridging and lay out practical ways of identifying and mitigating it. Specifically, we describe a simple fitting model that enables researchers to diagnose the severity of bridging artifacts, determine whether they can be minimized, and more accurately evaluate polyubiquitin-binding specificity.