Description
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. The Ubiquitin-Eu cryptate allows flexible assay development assessing each step in the ubiquitin pathway.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
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
