Determination of endogenous extracellular signal-regulated protein kinase by microchip capillary electrophoresis.
The application of microchip capillary electrophoresis (CE) to the assay of extracellular signal-regulated protein kinase (ERK) is presented. In this assay, ERK catalyzes the transfer of gamma-phosphate from adenosine 5(')-triphosphate to the threonine residue of a fluorescently labeled nonapeptide (APRTPGGRR), and the phosphorylated and nonphosphorylated peptides were detected by fluorescence. The phosphorylated and nonphosphorylated peptides and the internal standard were separated within 20s, and the increase in magnitude of the phosphorylated peptide peak was monitored to assess ERK activity. ERK reactions were prepared off-chip and analyzed on a single-lane glass microchip fabricated by standard methods. It was demonstrated that microchip CE could be used to measure endogenous amounts of ERK by spiking known concentrations of recombinant ERK2 into the lysates of serum-starved human umbilical vein endothelial cells (HUVEC) and recovering between 90 and 100% for all samples. Endogenous ERK activity was determined by microchip where HUVEC were stimulated with 500pM vascular endothelial growth factor (VEGF) at different times before cell lysis. The results showed a transient VEGF-mediated ERK activation that peaked at 10min, which was consistent with previous reports using conventional techniques. The microchip assay provided a rapid, accurate, and precise alternative to conventional methods of determining endogenous ERK activity.
Starkey
Dustin E
DE
Department of Chemistry, University of Cincinnati, OH 45221, USA.
Abdelaziez
Yasser
Y
Ahn
Chong H
CH
Tu
Jian
J
Anderson
LaShonda
L
Wehmeyer
Kenneth R
KR
Izzo
Nicholas J
NJ
Carr
Andrew N
AN
Peters
Kevin G
KG
Bao
James J
JJ
Halsall
H Brian
HB
Heineman
William R
WR
D002478:Cells, Cultured; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008832:Microchemistry; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis / Q000737:chemistry / Q000592:standards; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis* / Q000737:chemistry / Q000592:standards; D010766:Phosphorylation; D012015:Reference Standards; D015203:Reproducibility of Results; D013997:Time Factors; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
D042461:Vascular Endothelial Growth Factor A; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases