Affinity microfluidics enables high-throughput protein degradation analysis in cell-free extracts

Abstract

AbstractProtein degradation mediated by the ubiquitin-proteasome pathway regulates signaling events in many physiological and pathological conditions. In vitro degradation assays have been instrumental in the understanding of how cell proliferation and other fundamental cellular processes are regulated. These assays are direct, time-specific and highly informative but also laborious, typically relying on low-throughput polyacrylamide gel-electrophoresis followed by autoradiography or immunoblotting. We present protein degradation on chip (pDOC), a MITOMI-based integrated microfluidic technology for discovery and analysis of proteins degradation in cell-free extracts. The platform accommodates hundreds of microchambers on which protein degradation is assayed quickly, simultaneously and using minute amounts of reagents in one or many physiochemical environments. Essentially, pDOC provides a sensitive multiplex alternative to the conventional degradation assay, with relevance to biomedical and translational research associated with regulated proteolysis.