Ubiquitin assay toolbox

Ubiquitin toobox, a line of HTRF® reagents for monitoring protein ubiquitination.

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 reagents necessary for flexible assay development and scale up to HTS, assessing each step in the ubiquitin pathway.

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Assay Principle

Assay Theory

HTRF Ubiquitin assays use ubiquitin labeled with either Eu3+-Cryptate or biotin.

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 & the HTRF detection steps.

Enzymatic step: The target protein is incubated with the ubiquitin enzymes (E2 & E3) and Eu3+-labeled ubiquitin. The reaction starts with the addition of E1 enzyme.

Detection step: The ubiquitinated target protein is then detected by the addition of an XL665-labeled reagent that contains EDTA to stop the enzymatic reaction.

Outsource your ubiquitin assay

The Cisbio Bioassays team includes experts in ubiquitination assay development, and offers its outsourcing services for your specific targets.

Product Performance

p53 poly-ubiquitination monitoring

The figure 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.

The ubiquitin assay was performed following the procedure as described by Yabuki et al. in 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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Ubiquitin-Eu cryptate - 5,000 tests61UBIKLA
Ubiquitin-Eu cryptate - 20,000 tests61UBIKLB

Companion products

DescriptionCat. noProduct insertMSDS
Streptavidin-XL665 - 5,000 tests610SAXLA
Streptavidin-XL665 - 20,000 tests610SAXLB