High performance isoelectric focusing using capillary electrophoresis instrumentation.

High performance isoelectric focusing in capillaries provides rapid, high resolution separation of proteins based on their isoelectric points. Results can be obtained in a matter of minutes with little or no sample preparation. The technique requires the use of coated capillaries to reduce electroendosmosis so that stable, focused zones can be attained. Once focused, protein zones may be mobilized by the addition of salt to the catholyte or anolyte buffer. On-tube UV monitoring enables direct detection of sample components during mobilization with mass sensitivity equal to that of silver staining. The linear relationship between mobilization time and isoelectric point allows the technique to be used for estimation of protein pI. Demonstrated applications include separation of proteins in biological fluids with possible clinical applications, and characterization of biopharmaceutical proteins and monoclonal antibodies.