TY - JOUR
T1 - Native Protein Separation by Isoelectric Focusing and Blue Gel Electrophoresis-Coupled Two-Dimensional Microfluidic Chip Electrophoresis
AU - Hou, Ni
AU - Chen, Yu
AU - Yu, Shiyong
AU - Quan, Zongliang
AU - Pan, Chenhua
AU - Deng, Yulin
AU - Geng, Lina
N1 - Publisher Copyright:
© 2014, Springer-Verlag Berlin Heidelberg.
PY - 2014/9/28
Y1 - 2014/9/28
N2 - The use of microfluidic chip-based two-dimensional separation holds great promise in the proteomics field, given its portability, simplicity, speed, efficiency, and throughput. However, inclusion of sodium dodecyl sulfate, reported to be necessary for increasing protein-resolving capability, was also accompanied by the loss of both protein conformation and biological function. Here, we describe separation of native proteins by introducing blue native gel electrophoresis into isoelectric focusing and gel electrophoresis (IEF/CGE)-coupled protein two-dimensional microfluidic chip electrophoresis. After assessing the influence of various experimental conditions, the best separation ability and reproducibility of blue native IEF/CGE (IEF/BN-CGE) chip electrophoresis achieved until now were demonstrated no matter whether with a simple simulated mixture or with a complex mixture of total Escherichia coli proteins. Finally, instead of theoretical calculations, the image analysis technique was also used for the first time to quantitatively evaluate the actual peak capacities of chip electrophoresis. According to the number of features abstracted in the electrophoresis patterns, the superiority of the IEF/BN-CGE two-dimensional microfluidic chip electrophoresis was then exhibited quantitatively. The high native protein separation performance makes this established chip electrophoresis method possible for further application in widely needed drug screening, analysis of bio-molecular function, and assays of protein–protein interactions.
AB - The use of microfluidic chip-based two-dimensional separation holds great promise in the proteomics field, given its portability, simplicity, speed, efficiency, and throughput. However, inclusion of sodium dodecyl sulfate, reported to be necessary for increasing protein-resolving capability, was also accompanied by the loss of both protein conformation and biological function. Here, we describe separation of native proteins by introducing blue native gel electrophoresis into isoelectric focusing and gel electrophoresis (IEF/CGE)-coupled protein two-dimensional microfluidic chip electrophoresis. After assessing the influence of various experimental conditions, the best separation ability and reproducibility of blue native IEF/CGE (IEF/BN-CGE) chip electrophoresis achieved until now were demonstrated no matter whether with a simple simulated mixture or with a complex mixture of total Escherichia coli proteins. Finally, instead of theoretical calculations, the image analysis technique was also used for the first time to quantitatively evaluate the actual peak capacities of chip electrophoresis. According to the number of features abstracted in the electrophoresis patterns, the superiority of the IEF/BN-CGE two-dimensional microfluidic chip electrophoresis was then exhibited quantitatively. The high native protein separation performance makes this established chip electrophoresis method possible for further application in widely needed drug screening, analysis of bio-molecular function, and assays of protein–protein interactions.
KW - Blue gel electrophoresis
KW - Isoelectric focusing
KW - Native protein separation
KW - Peak capacity evaluation
KW - Two-dimensional microfluidic chip electrophoresis
UR - http://www.scopus.com/inward/record.url?scp=84919435442&partnerID=8YFLogxK
U2 - 10.1007/s10337-014-2728-3
DO - 10.1007/s10337-014-2728-3
M3 - Article
AN - SCOPUS:84919435442
SN - 0009-5893
VL - 77
SP - 1339
EP - 1346
JO - Chromatographia
JF - Chromatographia
IS - 19-20
ER -