Improved capillary electrophoresis conditions for the separation of kinase substrates by the laser micropipet system.
Phosphorylated and nonphosphorylated forms of peptide substrates for protein kinase C (PKC) and calcium-calmodulin activated kinase II (CamKII) were separated by capillary zone electrophoresis. Electrophoresis of the peptide substrates and products in biologic buffer solutions in uncoated capillaries yielded asymmetric analyte peaks with substantial peak tailing. Some of the peptides also exhibited broad peaks with unstable migration times. To improve the electrophoretic separation of the peptides, several strategies were implemented: extensive washing of the capillary with a base, adding betaine to the electrophoretic buffer, and coating the capillaries with polydimethylacrylamide (PDMA). Prolonged rinsing of the capillaries with a base substantially improved the migration time reproducibility and decreased peak tailing. Addition of betaine to the electrophoretic buffer enhanced both the migration time stability as well as the theoretical plate numbers of the peaks. Finally PDMA-coated capillaries brought about significant improvements in the resolving power of the separations. These modifications all utilized an electrophoretic buffer that was compatible with a living biologic cell. Consequently they should be adaptable for the new capillary electrophoresis-based methods to measure kinase activation in single cells.
Li
H
H
Department of Physiology & Biophysics, University of California, Irvine 92697-4560, USA.
Wu
H Y
HY
Wang
Y
Y
Sims
C E
CE
Allbritton
N L
NL
D000595:Amino Acid Sequence; D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000592:standards; D007834:Lasers; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D015203:Reproducibility of Results; D013379:Substrate Specificity
D002021:Buffers; D011493:Protein Kinase C