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 PROTAC on substrate ubiquitylation in several samples.
This Assay is both a service and an off-the-shelf kit. For more information about UbiTest, please also visit our UbiTEST product pages, where you can purchase the 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.
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
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.