Lumit™ protein interaction immunoassays using protein tags and biotinylated small molecules

Protein interactions including, protein-protein interactions (PPI) and protein-small molecule interaction, play critical roles in numerous cellular processes such as trafficking, signaling, apoptosis, and proliferation. As major drivers of signaling cascades, deregulated protein interactions are implicated in disease states including autoimmune diseases and cancers. Thus, modulating protein interactions is a major focus in drug discovery research. Efforts to screen for protein interaction inhibitors can be marred by technical limitations, as traditional immunoassays involve multiple wash steps and are not easy to adapt into a high throughput screening format. To overcome these limitations, we developed Lumit™ Protein Interaction Immunoassay using protein tags, a homogenous bioluminescent assay to measure protein-protein or protein-small molecule interactions in buffer. In these assays, streptavidin and antibodies against His, GST, FLAG®, and human-Fc are labeled with NanoBiT complementation reporter subunits LgBiT and SmBiT. When two proteins with different tags bind, the interaction can be detected by the complementation of SmBiT and LgBiT on corresponding antibodies. The simple add and read protocol enables fast, highly sensitive assays suitable for multiplexing and high throughput screens. Here, we demonstrate the utility of Lumit™ in several formats. First, we test how different small molecule inhibitors interfere with the interactions of KRAS mutants and RBD-cRAF interaction. Next, we monitor the interactions of tyrosine kinases with small molecule inhibitors. Finally, we demonstrate how Lumit can be used to monitor autoubiquitination of E3 ligase Cbl-b. Together, these data illustrate the utility of Lumit™ protein interaction immunoassay using protein tags and biotinylated small molecules for a variety of assays.