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

J Chromatogr A. 2004 Nov 12;1056(1-2):237-42.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Electrophoresis, Capillary / methods*
  • Green Fluorescent Proteins / metabolism*
  • Microscopy, Electron
  • Mitogen-Activated Protein Kinase 1 / metabolism*
  • Phosphorylation
  • Protein Transport
  • Proteins / metabolism*
  • Rats

Substances

  • Proteins
  • Green Fluorescent Proteins
  • Mitogen-Activated Protein Kinase 1