The proteasome is a multisubunit protein complex responsible for the degradation of proteins, making it essential in myriad cellular processes. Several reversible and irreversible peptide substrates inspired by known proteasome inhibitors have been developed to visualize it and monitor its activity; however, they have limited commercial availability or possess fluorophores that overlap with other known chemical probes, limiting their simultaneous use. The protocols presented here describe the synthesis of a clickable epoxomicin-based probe followed by the copper-catalyzed installment of an azide-containing fluorophore, and the application of the synthesized peptide in proteasome activity assays by SDS-PAGE and flow cytometry. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Solid-phase synthesis of clickable peptide fragment (2) Basic Protocol 2: In-solution coupling of epoxy-ketone moiety to fragment (2) Basic Protocol 3: Copper-catalyzed click reaction of (3) with fluorophore of choice Basic Protocol 4: Monitoring proteasome activity by SDS-PAGE in HEK-293T cells Alternate Protocol: Monitoring proteasome activity by flow cytometry in HEK-293T cells.
Keywords: cell-based assay; chemical probes; flow cytometry; in-gel fluorescence; proteasome.
© 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.