Although Biacore® instrumentation can be used to study many different binding reactions, it has limitations.
- As the instrument records changes in the angle of light reflected from the surface on which binding occurs, and those changes are dependent on very small, local changes in the refractive index of the solution, any experiments that use solutions with significantly different refractive indices (e.g., aqueous buffer vs buffer containing 10% glycerol) will create large, non-specific signals that are difficult to correct. Even small differences in refractive index can complicate quantitative analyses.
- Analysis of binding reactions of very high affinity should be interpreted with caution. Binding affinities of nM or better can be subject to an artifact known as “mass transfer,” in which the binding of the analyte to the ligand surface lowers the free concentration of analyte. As all analyses require accurate values for the free analyte concentrations, the values calculated when mass transfer is occurring are inaccurate. There are several ways to correct for this, including reducing the amount of ligand on the chip surface, increasing the rate of injection of analyte, and utilizing the mass transfer correction algorithm in the BiaSimulation software package.
- Mathematical fits of curves can provide values for rate constants and affinity binding constants, but they depend on a model of binding that cannot be tested by a Biacore® instrument alone. The quality of your results depends on the model you choose (e.g., 1:1 binding of analyte to ligand, 2:1, cooperative, etc.). Although BiaSimulation software allows you to test different models to optimize the process of curve fitting, the validity of the model used should be tested by other means.
- With rare exceptions, the Biacore® 3000 cannot be used to measure binding of molecules with molecular masses < 1.5 kDa. The Biacore® T200 should be used specifically for studies of binding of small molecules.
- Although Biacore® instruments can measure binding between molecules with apparent affinities ranging from subnanomolar to high micromolar, it cannot detect some binding reactions reliably, even when clear evidence for binding is obtained by other methods.