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.
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.
|Figure 2. Measuring ubiquitination of GFP–IkBalpha in response to agonist stimulation. |
A. The LanthaScreen™ format is based on the use of a long-lifetime Tb chelate as the donor species and fluorescein as the acceptor species. When Tb- and fluorescein-labeled molecules are brought into proximity, energy transfer takes place, causing an increase in acceptor fluorescence and a decrease in donor fluorescence. These fluorescent signals can be read in a timeresolved manner to reduce assay interference and increase data quality. B. The time-resolved spectrum illustrates energy transfer occurring when Tb and fluorescein are brought into proximity via biomolecular interactions. The TR-FRET value is determined as a ratio of the FRET-specific signal measured with a 520 nm filter to the terbium-specific signal measured with a 495 nm filter. The inset shows the time-resolved spectrum in the absence of energy transfer.
For Research Use Only. Not for use in diagnostic procedures.