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 ubiquitylated fraction simplifies comparisons between different samples, making this assay perfect for comparing the effect of small molecules/stimuli on substrate ubiquitylation in several samples.
For more a more in depth technological overview, visit our UbiTest Service page. Visit our UbiTest 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.