Ubiquitin Assay Toolbox
Ubiquitination plays a key role in regulating protein degradation and is carried out by the conjugation of ubiquitin to target proteins through the sequential activity of three enzymes: E1, E2 and E3. Poly-ubiquitinated proteins are then subject to degradation by proteasomes. The ubiquitin pathway is associated with many pathologies such as cancer, muscle atrophy, inflammation, viral infection, and metabolic and neurodegenerative disorders. Cisbio Bioassays offers a comprehensive line of HTRF® reagents for investigating ubiquitin pathway targets. The HTRF® Ubiquitin toolbox assembles all the necessary reagents for flexible assay development and scale up to HTS to assess each step in the ubiquitin pathway.
- Highly sensitive assay
- Low enzyme consumption
- Results within 1-2 hrs
- Stable signal > 24 hrs
- Miniaturization down to <10µL
- Detection of auto-, mono-, poly-ubiquitination
- Monitoring activity of each enzyme involved in ubiquitination ( E1, E2, or E3)
Assay Theory:
HTRF Ubiquitin assays use ubiquitin that is labeled with either Eu3+-Cryptate or biotin which is then recognized by the E1, E2, and E3 enzymes and incorporated into the growing poly-ubiquitin chain on the target protein. Cisbio Bioassays's toolbox reagents allow flexibility in the type of target protein used in the assay including biotinylated proteins, tag-labeled fusion proteins or native proteins recognized by an antibody. Upon the addition of XL665 conjugate, the HTRF signal is proportional to the level of ubiquitination and remains stable for more than 24 hours. An example of a p53-polyubiquitination assay is shown here.
Assay Principle:
HTRF ubiquitin assays have two basic phases: the enzymatic step followed by HTRF detection. During the enzymatic step the target protein is incubated with the ubiquitin enzymes (E1, E2 & E3) and Eu3+-labeled ubiquitin. The reaction starts with the addition of E1 enzyme. The ubiquitinated target protein is then detected by the addition of an XL665-labeled reagent (will depend on target protein type) that contains EDTA to stop the enzymatic reaction.
Assay Performance:
p53 poly-ubiquitination monitoring:
Figure 1 summarizes the results of a p53 poly-ubiquitination
assay. Eu3+-labeled ubiquitin was directly incorporated into the poly-ubiquitin chain on the biotinylated-p53
target protein . The ubiquitin reaction was stopped at various times by adding EDTA. Poly-ubiquitinated p53 was
measured by the addition of streptavidin-XL665. The enzymatic reaction was linear for up to 50 minutes.
The assay had CVs <5% and a S/B of 10 that was stable for several days.
Figure 1: The ubiquitin assay was performed following the procedure as described by Yabuki et al. Comb Chem High Throughput Screening. 1999; 2: 27-87. Briefly, biotinylated-p53 (200nM) was incubated with E2 (300nM), E3 (150nM), ATP (200mM), and a mixture of Ub-K/Ub (20/500 nM). E1 (8nM) was added to start the reaction and EDTA (12.5 mM) added at various times to stop the reaction.
Outsource your ubiquitin assay:
The Cisbio Bioassays team includes experts in ubiquitination assay development, and offers its outsourcing services for your specific targets.
Ordering Info:
Ubiquitin Assay Reagents:
| Description | Quantity* | Cat no. |
| Ubiquitin-Cryptate | 5,000 tests | 61UBIKLA |
| Ubiquitin-Cryptate | 20,000 tests | 61UBIKLB |
| Ubiquitin Assay Development | - | 63ADR000 |
| *based on 20 µl assay volume | ||
Companion Products:
| Description | Cat no. |
| Sa-XL665 | Streptavidins |
| Sa-XLent! | Streptavidins |
| Anti-6HIS-XL665 | Anti-Tag |
| Anti-GST-XL665 | Anti-Tag |
For more information or To Place an Order:
U.S. and Canada
1-888-963-4567
Europe and other countries
+33(0)466 796 705
Japan
+81-3-5510-2932
