Interferometry is one of several techniques that can be used to probe the concentration and binding interactions of biomolecules, provided that the molecule’s binding partner is immobilized on a surface. Bornhop et al. now show that a fairly simple realization of back-scattering interferometry, which makes use of microfluidic channels for sample mixing and as the multipath cell for the laser light, can be used to determine dissociation constants for several pairs, apparently through changes in refractive index upon binding. Dissociation constants were determined for several binding pairs, including protein A with immunoglobulin G and activated calmodulin (CaM) with a small-molecule inhibitor or with calcineurin. Unlike microcalorimetric methods, these assays can be performed with very low amounts of samples; for example, the several CaM assays required only 200 picomoles of this protein.
Free-solution, label-free molecular interactions can be investigated using back-scattering interferometry (BSI), which allows quantification of Kd values ranging from micromolar to picomolar using minuscule quantities of the binding pairs.