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

Koszela J, Pham NT, Evans D, Mann S, Perez-Pi I, Shave S, Ceccarelli DFJ, Sicheri F, Tyers M, Auer M

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.

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.

BMC Biol. 2018;16(1):88.

Pubmed ID: 30097011

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