Protein Characterization Facility: We have several different instruments to measure macromolecular interaction kinetics and thermodynamics. These comprise of three instruments that use Surface Plasmon Resonance (SPR) and two that use Biolayer Interferometry. The Facility is used by multiple faculty from across the Division as well as some external users, and have an occupancy rate of about 90% and very little down time. The instruments are heavily used because of their relatively small sample requirements and accurate measurements of binding affinities.
A) Surface Plasmon Resonance (SPR) is a non-invasive optical measuring technique which measures the mass concentration of biomolecules in close proximity to a specially prepared surface. The technique does not require any labeling of the interacting components. The response is largely independent of the nature of the biomolecule, so that all steps in an interaction analysis may be followed with the same detection technique. The process involves flow of one molecule (analyte) over a surface with an immobilized binding partner (ligand). Binding results in a change in refractive index at the surface which is transduced to an optical signal. Dissociation of bound analyte is monitored by flowing buffer over the surface.
Biacore 2000 and 3000 Systems are two very robust SPR instruments. These instruments use a single chip with four surfaces to which different molecules can be attached, making it possible to simultaneously determine 3 different affinities. We have recently acquired a ProteOn XPR36 array based system with funds from the DBT-IISc partnership program. This allows for one shot kinetics and can have upto 36 ligands immobilized on the chip surface and has greatly increasedthe throughput of the facility.
B) Biolayer interferometry (BI) This technology is another analytical technique with current applications in Surface Science and Biophysics. The instrument records thickness, mass, density, refractive index of the interfacially adsorbed layers. The physical dimensions of (protein) layers at the surface, can be measured, making it possible to determine molecular assembly/stoichiometry in addition to the kinetics and equilibrium constant for the reaction. We have two instruments that employ this technology, the older AnaLight® 4D and the recently acquired Octet Red96. The Octet has the advantage of negligible analyte consumption and low consumable costs and was procured very recently with funding from MHRD. The Octet system is ideally suited for 96-well characterization of protein-protein and protein-small molecule binding kinetics, and for the determination of protein concentrations and titer.