Improved capillary electrophoresis conditions for the separation of kinasesubstrates by the laser micropipet system

Phosphorylated and nonphosphorylated forms of peptide substrates for protei n kinase C (PKC) and calcium-calmodulin activated kinase II (CamKII) were s eparated by capillary zone electrophoresis. Electrophoresis of the peptide substrates and products in biologic buffer solutions in uncoated capillarie s yielded asymmetric analyte peaks with substantial peak tailing. Some of t he peptides also exhibited broad peaks with unstable migration times. To im prove the electrophoretic separation of the peptides, several strategies we re implemented: extensive washing of the capillary with a base, adding beta ine to the electrophoretic buffer, and coating the capillaries with polydim ethylacrylamide (PDMA). Prolonged rinsing of the capillaries with a base su bstantially improved the migration time reproducibility and decreased peak tailing. Addition of betaine to the electrophoretic buffer enhanced both th e migration time stability as well as the theoretical plate numbers of the peaks. Finally PDMA-coated capillaries brought about significant improvemen ts in the resolving power of the separations. These modifications all utili zed an electrophoretic buffer that was compatible with a living biologic ce ll. Consequently they should be adaptable for the new capillary electrophor esis-based methods to measure kinase activation in single cells. (C) 2001 E lsevier Science B.V. All rights reserved.