Publication — IRIC

Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay.

The ubiquitin-proteasome system (UPS) controls the stability, localization and/or activity of the proteome. However, the identification and characterization of complex individual ubiquitination cascades and their modulators remains a challenge. Here, we report a broadly applicable, multiplexed, miniaturized on-bead technique for real-time monitoring of various ubiquitination-related enzymatic activities. The assay, termed UPS-confocal fluorescence nanoscanning (UPS-CONA), employs a substrate of interest immobilized on a micro-bead and a fluorescently labeled ubiquitin which, upon enzymatic conjugation to the substrate, is quantitatively detected on the bead periphery by confocal imaging.

Publication date
August 10, 2018
Principal Investigators
Koszela J, Pham NT, Evans D, Mann S, Perez-Pi I, Shave S, Ceccarelli DFJ, Sicheri F, Tyers M, Auer M
PubMed reference
BMC Biol. 2018;16(1):88
PubMed ID
30097011
Affiliation
Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, University of Edinburgh, C H Waddington Building, 3.07, Max Born Crescent, Edinburgh, EH9 3BF, UK. joanna.koszela@ed.ac.uk.