Real-Time Binding Studies Using Viable Cells

The binding of proteins to their target molecules is of interest to anyone studying biological processes, from cellular function and organism development, through to human disease and drug discovery. Several approaches exist to investigate and measure protein binding, including ELISA, Western blot, isothermal titration calorimetry, KinExa, Surface Plasmon Resonance (SPR) and flow cytometry.

While each approach has its advantages and disadvantages, many suffer from technical limitations that impact on the biological insight they can provide. For example, many traditional methods involve labelling a protein of interest with radioisotopes or fluorescent tags, so that binding events can be tracked and measured. However, such labels may inhibit normal protein-target interactions in vitro, calling into question how well these data represent normal interactions in vivo.  Also, for many of these methods, the reaction being investigated must be allowed to reach equilibrium before reliable binding kinetics can be ascertained. This means that experiments can be lengthy to perform and that the temporal resolution provided may not be fine enough for many applications.

To circumvent this argument, many methods have been developed that do not require the use of labels. For example, SPR utilises an optical approach to detect binding events, without the need for any labelling, by measuring changes in refractive index.  As SPR uses light to detect binding events, it also requires that the sample mixture passing over the sensor be relatively simple and clean. For this reason, SPR can only be used with purified receptors and artificial membrane fractions, whereas deeper biological insight would be provided by being able to work with intact, viable cells.