Ubiquitin assay toolbox

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.

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.