LifeSensors’ High Throughput (HT) UbiTest is a customized medium-throughput assay for absolute quantification of ubiquitylated substrate levels in cells that combines UbiTest principles and TR-FRET technology.

Conceptual schematic of the HT-UbiTest assay. Ubiquitylated proteins are enriched and isolated by Magnetic TUBEs, then eluted from the TUBEs. The isolated polyubiquitylated eluate is then digested with a broad-spectrum DUB to remove ubiquitin chains.  Finally, the protein of interest is detected in the digested polyubiquitlyated eluate using a homogenous two-antibody detection system based on either TR-FRET or AlphaLISA technology.


Sample data from the HT-UbiTest assay:
 MM.1S cells were treated with DMSO or BTZ. After treatment and cell lysis, p53 ubiquitylation was quantified using the p53 HT-UbiTest assay. In parallel, an immunoblot-based UbiTest was performed, which confirmed the results of the HT-UbiTest.

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