Ubiquitin Biomarker Assays
Ubiquitin Biomarker Assays
Determining the ubiquitylation state of proteins in cells is critical for understanding the endogenous mechanisms of the ubiquitylation cascade. Furthermore, assays that measure cellular ubiquitlyation enable the monitoring of compound treatment and biological stimulation. UbiQuant™ and UbiQuant S™ are ELISA-based assays for high-throughput determination of cellular ubiquitylation. For information about our high-throughput cellular assays,
The UbiTest platform replaces traditional immunoblotting methods for measuring cellular ubiquitylation, enabling simple comparisons of substrate ubiquitylation levels between samples and determination of ubiquitin chain linkage types. For more information about UbiTest.
Measuring cellular ubiquitylation in a plate-based format accelerates the process of ubiquitin pathway drug discovery. We have developed several platforms for high-throughput ubiquitylation that enable rapid, quantitative monitoring of cellular ubiquitylation.
LifeSensors’ UbiQuant™ ELISA is intended for the accurate determination of the concentration of total ubiquitin (poly- + mono-) in cell and tissue lysates. The kit is designed for research use only and is not intended for human or animal diagnostic or therapeutic applications.
LifeSensors’ UbiQuant™ S ELISA is intended for the relative determination of the concentration of a specific ubiquitylated, target protein in cells. This assay is designed to replace more laborious, semi-quantitative immunoprecipitation and Western blots to examine changes in ubiquitylation.
LifeSensors’ p53 High Throughput (HT) UbiTest is a customized assay for absolute quantification of ubiquitylated p53 levels in cells that combines UbiTest and TR-FRET technology.
Lots of key biomarkers are modified by ubiquitylation under different stimulation. The traditional method to determine protein of interest (POI) ubiquitylation depends on the smeared pattern of POI in the immunoblot after immunoprecipitation, which is generally considered unreliable and not quantitative. The smeared signal, while often indicative of a mixture of polyubiquitylated proteins, may also indicate other post-translational modifications such as SUMOylation, Neddylation or phosphorylation. Conversely, the absence of such a smear does not prove a lack of ubiquitylation. The smear may be very light or undetectable due to a dilutive effect: minimal amounts of a diverse mixture of polyubiquitylated species may be present. Furthermore, some antibodies may not robustly detect ubiquitylated forms of the POI because the epitope is masked by the conjugated ubiquitin molecule(s). LifeSensors,Inc, has developed a novel assay, UbiTest, which combines TUBE pull-down and DUB digestion, providing a more sensitive and reliable method for detecting endogenous POI ubiquitylation than either technique alone. In the UbiTest assay, non-linkage-selective TUBE is used to pull down all the polyubiquitylated proteins from the cell lysates. After elution of proteins from TUBE, the sample is split in half. One portion is treated with a broad spectrum DUB, which hydrolyzes polyubiquitin chains of all linkages. The DUB-treated portion is compared to the untreated portion using immunoblot for the POI. The appearance or increase of unmodified POI signal in the DUB-treated sample indicates that the POI is polyubiquitylated. This tool provides an efficient platform to unveiling the roles of ubiquitylation in essential pathways and decrypting the cellular consequences of the intricate ubiquitin code.