Direct monitoring of the expression of the green fluorescent protein-extracellular signal-regulated kinase 2 fusion protein in transfected cells using capillary electrophoresis with laser-induced fluorescence detection.
Proper subcellular localization of the extracellular signal-regulated kinases (ERKs) is important in regulating physiological functions such as proliferation and differentiation in the pheochromocytoma cell line (PC12 cells). Thus, a direct visualization method is necessary to observe ERK localization within the cell or in crude cellular extracts. In this paper, a determination method was established for the detection of ERK2 localization in PC12 cells using green fluorescent protein (GFP) and capillary electrophoresis with laser-induced fluorescence (LIF). GFP as a reporter or labeling tag for gene expression in biochemistry and cell biology was used for the detection of ERK2 localization in PC12 cells. PC12 cells were transfected with GFP-ERK2 plasmid construct that was inserted into a variant GFP gene (enhanced green fluorescent protein), and successfully expressed GFP-ERK2 fusion proteins. GFP-ERK2 fusion proteins were detected within 5 min by CE analysis using an uncoated fused-silica capillary with LIF. Optimum conditions for GFP-ERK2 fusion proteins detection were 100 mM 3-(cyclohexylamino)-1-propanesulfonic acid buffer containing 100 mM sodium dodecylsulfate, pH 11, running at 20 degrees C. This result offers new opportunity in screening for the determination of localization of intracellular components, protein-protein interactions and kinase activity within the cells.
Yoon
Seunghee
S
Ban
Eunmi
E
Yoo
Young Sook
YS
D000818:Animals; D015153:Blotting, Western; D049452:Green Fluorescent Proteins; D007834:Lasers; D008164:Luminescent Proteins / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D016716:PC12 Cells; D010957:Plasmids; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014162:Transfection
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019950:Mitogen-Activated Protein Kinase 1