In vitro SCFbeta-Trcp1-mediated IkappaBalpha ubiquitination assay for high-throughput screen.

An increasing body of evidence indicates that constitutive activation of NF-kappa B contributes to tumorigenesis and inflammation. Ubiquitination and degradation of I kappa B plays an essential role in NF-kappa B activation. Here we describe an in vitro I kappa B alpha ubiquitination assay system in which purified E1, E2, SCF beta-Trcp1 E3, I kappa B alpha, IKK2, and Ub were used to generate ubiquitinated I kappa B alpha. The ubiquitination of I kappa B alpha is strictly dependent on its phosphorylation by IKK2, as well as the presence of E1, E2, E3, and Ub. The assay was adapted into 384-well plate format in which an antibody against I kappa B alpha was used to capture I kappa B alpha, and the biotinylated ubiquitin attached to I kappa B alpha was detected with europium(Eu)-labeled streptavidin. This assay can be used to discover inhibitors of I kappa B alpha ubiquitination. Such inhibitors would block NF-kappa B activation by stabilizing I kappa B levels in cells and thus provide a new therapeutic approach to NF-kappa B-related human diseases.