Profiling Sodium-Dependent Phosphate Transporter NaPi-IIb with Resonant Waveguide Grating Biosensor
The development of label-free resonant waveguide grating (RWG) technology in microplate format in the last decade has stimulated interests from academic and industrial scientists, leading to evaluation of the technology in a broad range of biochemical and cell-based assays. In cellular assays the plasma membrane and immediate cellular volume are within the sensing region from the surface of the RWG biosensor. Any rearrangement of intracellular proteins or molecules in this region of the cells, termed dynamic mass redistribution (DMR), can be detected by the RWG biosensor. The biosensor monitors a global cellular output that can amplify the activity of a few active receptors into a measurable response; therefore, it is a useful tool for measuring physiological responses of live cells.
Most membrane transporter studies still rely on measurement of the accumulation of radiolabeled substrates or on using electrophysiology techniques. Thus, there is an unmet need for a homogeneous and high-throughput assay for transporter research. The biological process of transporting substrates across the plasma membrane of cells and their subsequent interactions with intracellular proteins or molecules presents an ideal case study for the RWG biosensor. In this report, we describe a detailed protocol using an RWG biosensor to monitor DMR signals in cells following the activation of the type IIb sodium-dependent phosphate transporter (NaPi-IIb) in a homogeneous, 384-well assay format. Results suggest that the DMR signals could serve as novel and quantifiable physiological responses of activated NaPi-IIb transporters and the technology can be used to query transporter pharmacology.
Key wordsCellular assay Dynamic mass redistribution (DMR) High-throughput Label-free NaPi-IIb Resonant waveguide grating (RWG) Transporter
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