Determination of protein phosphorylation and the translocation of green fluorescence protein-extracellular signal-regulated kinase 2 by capillary electrophoresis using laser induced fluorescence detection

Abstract

In this study, we developed a method to monitor the phosphorylation and translocation of the extracellular signal-regulated kinase (ERK2) proteins after PC12 cells have been stimulated by a mitogen. The method involves the use of green fluorescent protein (GFP), capillary electrophoresis and the measurement of laser-induced fluorescence (CE-LIF). We showed the prescence of the non-phosphorylated GFP-ERK2 and phosphorylated GFP-ERK2 in cell lysates by CE-LIF, and then compared the phosphorylations of GFP-ERK2 and GFP-183A. Phosphorylated GFP-ERK2 was detected at 6.7 min and the non-phosphorylated GFP-ERK2 at 5.3-5.5 min. The results were compared with confocal laser scanning microscope imaging and western blot results, and suggest that the developed method can be used to detect other enzymatic modifications. (C) 2004 Elsevier B.V. All rights reserved.