Assay principle

HTRF Ubiquitin assays use ubiquitin labeled with Europium cryptate. 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 protocol

HTRF ubiquitin assays have two basic phases: the enzymatic step, followed by the HTRF detection step. 1. 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. 2. 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 assay

The figure on the right 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:

200 nM Biotinylated-p53 was incubated with 300nM E2, 150nM E3, 200nM ATP, and a mixture of Ub-K/Ub (20/500 nM).

8 nM E1 was then added to start the reaction,

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 Oct;2(5):279-87