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Rapid Real‐Time Ubiquitination Assays as Alternatives to Western Blotting
Author(s) -
de Silva Ruvindi,
Page Richard C.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04631
Subject(s) - ubiquitin , ubiquitin ligase , blot , hsp70 , western blot , microbiology and biotechnology , biology , chemistry , heat shock protein , biochemistry , gene
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 H sp70 I nteracting P rotein) 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 Information NIGMS R35 GM128595