Determination of protein phosphorylation and the translocation of green fluorescence protein-extracellular signal-regulated kinase 2 by capillary electrophoresis using laser induced fluorescence detection.
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.
Yoon
Seunghee
S
Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Korea.
Han
Kyoung-Youn
KY
Nam
Hui-Sun
HS
Nga
Le Viet
le V
Yoo
Young Sook
YS
D000818:Animals; D015153:Blotting, Western; D019075:Electrophoresis, Capillary / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism*; D051381:Rats
D011506:Proteins; D049452:Green Fluorescent Proteins; D019950:Mitogen-Activated Protein Kinase 1