Application of multiplexed capillary electrophoresis with laser-induced fluorescence (MCE-LIF) detection for the rapid measurement of endogenous extracellular signal-regulated protein kinase (ERK) levels in cell extracts.
Multiplexed (96-lane) capillary electrophoresis with laser-induced fluorescence (MCE-LIF) detection was used for the rapid analysis of extracellular signal-regulated protein kinase (ERK) levels from in vitro cell extracts. The levels of ERK enzyme in cell extracts were determined by monitoring the conversion of a fluorescent-labeled peptide substrate to a phosphorylated fluorescent-labeled peptide product using MCE-LIF. The incorporation of a fluorescent internal standard was found to improve the precision of the analysis. The enzyme assay conditions including substrate concentration, reaction time and enzyme linear range were rapidly optimized using the MCE-LIF approach for both direct and immunoprecipitation-based ERK assays. The levels of ERK from in vitro cell extracts stimulated with angiopoietin 1 (Ang1*) were determined using the MCE-LIF approach. The advantages of MCE-LIF for developing and applying enzyme assays, as well as the figures of merit for the direct and immunoprecipitation ERK assays, are discussed.
Procter & Gamble Pharmaceuticals, 8700 Mason-Montgomery Rd., Mason, OH 45040, USA.
Jian
J
Tu
LaShonda N
LN
Anderson
Jian
J
Dai
Kevin
K
Peters
Andrew
A
Carr
Paula
P
Loos
Danielle
D
Buchanan
James J
JJ
Bao
Changsheng
C
Liu
Kenneth R
KR
Wehmeyer
D002457:Cell Extracts; D002478:Cells, Cultured; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007700:Kinetics; D007834:Lasers; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis*; D011233:Precipitin Tests; D012015:Reference Standards; D013050:Spectrometry, Fluorescence / Q000379:methods*
D002457:Cell Extracts; D020928:Mitogen-Activated Protein Kinases