Rapid Real‐Time Ubiquitination Assays as Alternatives to Western Blotting

Abstract

The refolding and degradative pathways of the protein quality control system converge through the chaperoned ubiquitination complex formed by the interaction of the E3 ubiquitin ligase CHIP ( C‐terminus of Hsp70 Interacting Protein) and the ATP‐dependent chaperone Hsp70 (70 kilodalton heat shock protein). We have applied the CHIP/Hsp70 chaperoned ubiquitination system as a test for our newly developed ubiquitination assay, presenting a viable alternative to traditional western blots for detection of ubiquitination. Here we describe the cutting‐edge biolayer interferometry ubiquitination assay to rapidly assay ubiquitination in real time. Our BLI ubiquitination assay is recorded with 1‐second resolution and is the first assay to allow for determination of rate and extent of ubiquitination in real‐time. This assay detects formation of polyubiquitin chains directly on a biosensor‐bound target. Polyubiquitination of substrates using wild‐type components produces a robust signal. Reagent concentrations and quantities are similar to those used by traditional western blot‐based ubiquitination assays. Results are returned instantaneously and result in substantial time savings.Support or Funding InformationNIGMS R35 GM128595