Program on Apoptosis and Cell Death Research

Anderson, United States

Program on Apoptosis and Cell Death Research

Anderson, United States

Time filter

Source Type

Madiraju C.,Program on Apoptosis and Cell Death Research | Welsh K.,Program on Apoptosis and Cell Death Research | Cuddy M.P.,Program on Apoptosis and Cell Death Research | Godoi P.H.,Program on Apoptosis and Cell Death Research | And 8 more authors.
Journal of Biomolecular Screening | Year: 2012

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z′ scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13. © 2012 Society for Laboratory Automation and Screening.

Loading Program on Apoptosis and Cell Death Research collaborators
Loading Program on Apoptosis and Cell Death Research collaborators