김태욱
2017-09-04T01:30:22Z
2017-09-04T01:30:22Z
2015-11
BIOSENSORS & BIOELECTRONICS, v. 73, Page. 93-99
0956-5663
1873-4235
http://linkinghub.elsevier.com/retrieve/pii/S0956566315301421
http://hdl.handle.net/20.500.11754/28846
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.
This work was supported by Mid-career Researcher Program (Nos. 2013R1A2A2A03015161 and 2013R1A2A2A01014234) and Nano Material Technology Development Program (No. 2012M3A7B4035286) through the National Research Foundation (NRF) funded by the Ministry of Science, ICT, and Future Planning (MSIP). This work was also supported by Basic Science Research Program (No. 2012R1A6A1029029) through the NRF funded by the Ministry of Education.
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ELSEVIER ADVANCED TECHNOLOGY
Sequential phosphorylation
Microfluidic
Isoelectric focusing
Protein kinase
Inhibition assay
Immunoprecipitation
Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis
Article
73
10.1016/j.bios.2015.05.047
93-99
BIOSENSORS & BIOELECTRONICS
Choi, Hoseok
Choi, Nakchul
Lim, Butaek
Kim, Tae-Wuk
Song, Simon
Kim, Young-Pil
2015001548
S
COLLEGE OF NATURAL SCIENCES[S]
DEPARTMENT OF LIFE SCIENCE
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