E2 Selection

The  E2 SELECT™ Profiling & Selection Kit has been developed for the exploration of  functional competency between E2’s and E3’s.

At the core of the assay are microtiter plates pre-coated with a proprietary reagent that captures polyubiquitin chains formed in an E3 ligase-dependent reaction.

Principle

For the assay, an E1, E2, Ubiquitin (Ub) cocktail is present in each well of the plate, different wells containing different E2’s (with each of 24 human E2’s being represented by triplicate wells (see map, page 5).
 
An E3 ligase of interest, together with ATP, is then added directly to the wells. During the reaction, polyubiquitin chains generated by the E1-E2-E3 machinery are recognized and captured in the wells. Following the reaction and subsequent wash steps, the isolated poly-ubiquitylated product is incubated with Detection Reagent 1 and streptavidin-HRP allowing for detection by chemiluminescence. Thus, the signal generated by captured poly-ubiquitylated product in this "Sandwich ELISA-like assay" is a quantitative measure of E2-E3.
 
This detection strategy does not require additional non-native tagging or labeling of ubiquitin, which could lead to experimental artifacts. 

Benefits

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