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Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis

Title
Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis
Author
김태욱
Keywords
Sequential phosphorylation; Microfluidic; Isoelectric focusing; Protein kinase; Inhibition assay; Immunoprecipitation
Issue Date
2015-11
Publisher
ELSEVIER ADVANCED TECHNOLOGY
Citation
BIOSENSORS & BIOELECTRONICS, v. 73, Page. 93-99
Abstract
We report a simple method for analyzing sequential phosphorylation by protein kinases using fluorescent peptide substrates and microfluidic isoelectric focusing (mu IEF) electrophoresis. When a dye-labeled peptide substrate was sequentially phosphorylated by two consecutive protein kinases (mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3)), its differently phosphorylated forms were easily separated and visualized by fluorescent focusing zones in the WEE channel based on a change in the isoelectric point (pI) by phosphorylation. As a result, ratiometric and quantitative analysis of the fluorescent focusing regions shifted by phosphorylation enabled the analysis of phosphorylation efficiency and the relevant inhibition of protein kinases (MAPK and GSK3) with high simplicity and selectivity. Furthermore, the GSK3 activity in the cell lysates was elucidated by mu IEF electrophoresis in combination with immunoprecipitation. Our results suggest that this method has great potential for analyzing the sequential phosphorylation of multiple protein kinases that are implicated in cellular signaling pathways. (C) 2015 Elsevier B.V. All rights reserved.
URI
http://linkinghub.elsevier.com/retrieve/pii/S0956566315301421http://hdl.handle.net/20.500.11754/28846
ISSN
0956-5663; 1873-4235
DOI
10.1016/j.bios.2015.05.047
Appears in Collections:
COLLEGE OF NATURAL SCIENCES[S](자연과학대학) > LIFE SCIENCE(생명과학과) > Articles
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