In Vivo Validation and Biomarker Assays
As a LifeSensors customer, your in vivo validation process, focusing on high-throughput assay development, begins with a consultation with members of our team. You will discuss target(s), project goals, assay format, and assay parameters. The consultation will determine how experiments such as cell culture, assay development, technology transfer, and drug treatment will be divided among the LifeSensors and you as a customer. After project completion, customer deliverables may include any or all of the following: assay development summary, sample proof-of-concept data, assay SOP, assay reagents, or drug activity data (e.g. cellular IC50s). Assay reagents may be off-the-shelf products or customized products to be sold exclusively to you as a customer. The project timelines for this process range from 4-8 weeks depending on assay format, target selection, and goals. LifeSensors will work with you to generate a timeline estimate for your individual project process.
By collaborating with LifeSensors for your drug discovery needs, you will get the data you need to validate your best compounds for advancement from in vitro hits to animal and clinical studies.
As a LifeSensors customer, we will schedule a consultation with you to discuss target(s), project goals, antibody selection, control samples and experimental conditions, for your UbiTest order. The consultation will determine whether cell culture and treatment may need to be performed either by a LifeSensors team member, or whether you will provide LifeSensors with treated cell samples to analyze. A LifeSensors scientist will then perform UbiTest assays here in our laboratory. Post-project deliverables will include western blot data indicating the ubiquitylation level in each of your samples. Project time estimation ranges are based on the number of samples to be analyzed .
1-2 weeks for 1-6 samples
2-3 weeks for 7-12 samples
For samples greater than 13, you must contact us for an estimated project time line
Your final UbiTest report will show the relative level of your protein’s ubiquitylation across each sample you submit for analysis. This will allow you to determine the downstream effect of inhibitors or biological stimuli on the ubiquitylation of your protein of interest. While ubiquitin Western blots are challenging, our data reports will allow you to draw convincing conclusions and accelerate your research.
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.