In Vivo Validation and Biomarker Assays
In addition to enabling in vitro ubiquitin drug discovery, LifeSensors has the expertise to determine compound activity in a cellular context. Our assays for monitoring cellular ubiquitylation will help you quickly demonstrate the downstream effects of your E3 ligase or DUB inhibitor. Ranging in throughput from immunoblot to 384-well formats, our assay concepts can be readily adapted for a vast range of substrates on a custom basis. Thus, our technologies will enable the advancement of your compounds from in vitro lead compounds to in vivo and preclinical models.
High-Throughput Cellular Ubiquitylation Assays
Measuring cellular ubiquitylation in a plate-based format accelerates the process of ubiquitin pathway drug discovery. We have developed several platforms that enable rapid, quantitative monitoring of cellular ubiquitylation.
LifeSensors’ UbiQuant™ S ELISA and AlphaLISA assays are industry-leading platforms for measuring ubiquitylation of a target protein in cells. These assays utilized TUBEs (Tandem Ubiquitin Binding Entities) to detect ubiquitylated substrates, and are individually optimized for a specific substrate protein.
Conceptual schematic of the UbiQuant S ELISA assay. Polyubiquitlyated proteins are captured by an immobilized ubiquitin affinity matrix. Subsequently, unbound proteins are washed away, and ubiquitylated protein of interest bound to the matrix is identified using a POI-specific antibody and traditional ELISA detection reagents.
Conceptual schematic of the UbiQuant S AlphaLISA assay. TUBEs conjugated to AlphaLISA donor beads bind to polyubiquitylated proteins from a cell lysate. Simulataneously, POI antibodies conjugated to AlphaLISA acceptor beads bind to the POI in the cell lysate. If ubiquitylated POI is present, the concurrent binding of antibody and TUBE brings the AlphaLISA donor and acceptor beads into close proximity, resulting in a luminescent signal that is quantitatively correlated to the level of ubiquitlyated POI.
Sample data from UbiQuant S ELISA and AlphaLISA assays. Jurkat cells were treated with bortezomib (BTZ) or BTZ in combination with selective USP7 inhibitor P0217564 (564). Following treatment and cell lysis, Ubiquitylation of USP7 was measured by the UbiQuant S ELISA and AlphaLISA assays.
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.
LifeSensors’ UbiQuant™ ELISA is intended for the accurate determination of the concentration of total ubiquitin (poly- + mono-) in cell and tissue lysates. This assay is useful for monitoring the effects of broad-spectrum compounds such as proteasome inhibitors on total ubiquitin levels.
Conceptual schematic of the UbiQuant ELISA assay. Ubiquitlyated proteins are captured by an immobilized ubiquitin affinity matrix. Subsequently, unbound proteins are washed away, and total free ubiquitin and ubiquitin conjugates are quantified using an anti-ubiquitin antibody and traditional ELISA detection reagents.
Sample data from the UbiQuant ELISA assay. A serial dilution of ubiquitin was analyzed by the UbiQuant ELISA assay.
UbiTest: Simple Assays for Cellular Ubiquitylation
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. UbiTest is based on TUBE technology for enriching polyubiquitylated proteins. By visualizing the ubiquitylated fraction both with and without DUB digestion of polyubiquitin chains, UbiTest streamlines the confirmation of ubiquitylation status.
Previously, ubiquitin immunoblots have relied on the visualization of a qualitative, often deceptive smearing pattern, but no longer! Furthermore, the single band in the DUB-treated ubiquitlyated fraction simplifies comparisons between different samples, making this assay perfect for comparing the effect of biological stimuli or compound treatments on substrate ubiquitylation in several samples.
For more information about UbiTest, please also visit our product pages, where you can purchase off-the-shelf assay kits for performing UbiTest in your own lab.
Conceptual schematic of the UbiTest assay. Ubiquitylated proteins are enriched and isolated by Magnetic TUBEs, then eluted from the TUBEs. The isolated polyubiquitylated eluate is then split into two fractions: one fraction is left untreated, while the second fraction is digested with a broad-spectrum DUB to remove ubiquitin chains. Both fractions are analyzed by immunoblot using an antibody for the POI. The removal of the ubiquitin chains from the polyubiquitylated POI results in the simple visualization of a single, unmodified band of POI in the immunoblot.
Sample data from the UbiTest assay. Human PBMCs were stimulated with CD3/CD28 antibodies for 0-30 minutes. Cells were lysed to stop treatment and lysates were analyzed by UbiTest assay. The DUB+ lane for each time point clearly indicates an increase in ZAP70 ubiquitylation over time.