SPR Microscopy

Unlike soluble proteins, membrane proteins such as GPCRs, ion channels, solute transporters, etc... are difficult to purify, and are not typically amenable to traditional biophysical techniques for characterization of ligand affinities and kinetics.  Often, they must be extracted and purified in harsh detergents, which can affect their biochemical and biophysical properties.

SPR microscopy offers a solution to this problem by evaluating ligand affinities and kinetics for membrane targets in their native membrane environment in intact cells.  Since no purification of the target is required, SPRm allows for rapid evaluation of small and large molecules.  Unlike other cell-based biophysical methods, SPRm offers the ability to monitor ligand binding in a label-free manner, using either attached or suspension cells.

Unlike conventional SPR, which uses a homogeneous layer of purified protein immobilized on a gold surface, SPRm utilizes whole cells (live or fixed) which are grown or immobilized onto the chip surface.  Binding of a ligand causes micro-deformations in the cell membrane, which ultimately result in a SPR signal.  After a field of view with sufficient cell density is selected by the user, ligand is introduced through the sealed flow cell, and the SPR signal is monitored at multiple regions of interest, or ROIs.  These ROIs can be considered independent channels, and the software automatically performs the necessary filtering and background correction to analyze the 100s of resulting sensorgrams.  We and many others have shown that the binding kinetics and affinities from SPRm compare extremely well with data from other, more laborious methods such as radioligand binding assays.

The sensitivity of the Biosensing Instruments SPRm200 allows for accurate detection of binding of small molecules to membrane proteins.  Click "Learn More" below for an example of a 400 Da small molecule binding to a membrane transporter in HEK293 cells.

The application note below compares the kinetics and affinities of several different ligands to their cognate membrane protein targets in both live and fixed cells, showing that cell fixation does not significantly alter binding.

More information on SPRm technology as well as many more examples can be found on the Biosensing Instruments website.