Cellular Lysate Proteasome Assays
Difficulties inherent in recreating relevant biological models within the confines of a microtiter plate often limit early discovery efforts to simplified assay systems using purified components. Such models cannot interrogate your target within its complex cellular environment. Despite this key shortcoming, biochemical assays are often preferred over cell-based assays due to perceived differences in complexity, time, and cost.
Our LanthaScreen™ Cellular Assays detect posttranslational modifications, such as ubiquitination of specific target proteins that take place within the native cellular environment. By expressing the protein of interest as a fusion with GFP, a modification-specific antibody for ubiquitin labeled with the TR-FRET donor partner terbium can be used to quantitatively detect these modifications following cell lysis (Figure 1), providing a higher throughput alternative to methods such as western blots or ELISAs.
Figure 1. Schematic of the LanthaScreen™ cellular assay format.
Cellular Ubiquitination Assay Case Study: NFkappaB Pathway
Activation of the NFkappaB pathway induces a signal transduction cascade that results in phosphorylation, ubiquitination, and proteasomal degradation of IkappaBalpha. Upon IkappaBalpha degradation, liberated NFkappaB translocates to the nucleus to activate target gene expression. Using a clonal cell line expressing GFP–IkappaBalpha, we have developed a cellular assay capable of measuring TNFalpha–dependent ubiquitination (Figure 2) of endogenously expressed IkappaBalpha. This technology provides a powerful means to interrogate the intermediate steps in NFkappaB signaling, without compromising the endogenous physiological complexity of the signaling pathway.