Reciprocating free-flow isoelectric focusing device for preparative separation of proteins.
The traditional recycling free-flow isoelectric focusing (RFFIEF) suffered from complex structure, tedious operations and poor extensibility as well as high cost. To address these issues, a novel reciprocating free-flow isoelectric focusing device (ReFFIEF) was developed for proteins or peptides pre-fractionation. In the new device, a reciprocating background flow was for the first time introduced into free flow electrophoresis (FFE) system. The gas cushion injector (GCI) used in the previous continuous free-flow electrophoresis (CFFE) was redesigned for the reciprocating background flow. With the GCI, the reciprocating background flow could be achieved between the GCI, separation chamber and transient self-balance collector (tSBC). In a run, process fluid flowed to and from, forming a stable reciprocating fluid flow in the separation chamber. A pH gradient was created within the separation chamber, and at the same time proteins were focused repeatedly when passing through the chamber under perpendicular electric field. The ReFFIEF procedure was optimized for fractionations of three model proteins, and the optimized method was further used for pre-fractionation of model human serum samples. As compared with the traditional RFFIEF devices developed about 25 years ago, the new ReFFIEF system showed several merits, such as simple design and structure, user-friendly operation and easy to extend as well as low cost.
Laboratory of Analytical Biochemistry and Bioseparation, State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Fan-Zhi
FZ
Kong
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
Ying
Y
Yang
Laboratory of Analytical Biochemistry and Bioseparation, State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Yi
Y
Wang
Laboratory of Analytical Biochemistry and Bioseparation, State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Guo-Qing
GQ
Li
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
Shan
S
Li
Laboratory of Analytical Biochemistry and Bioseparation, State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Hua
H
Xiao
Laboratory of Analytical Biochemistry and Bioseparation, State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, United States.
Liu-Yin
LY
Fan
Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, United States. Electronic address: shaorong.liu@ou.edu.
Shao-Rong
SR
Liu
Laboratory of Analytical Biochemistry and Bioseparation, State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: cxcao@sjtu.edu.cn.
Cheng-Xi
CX
Cao
D001798:Blood Proteins / Q000032:analysis / Q000302:isolation & purification; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004586:Electrophoresis / Q000295:instrumentation*; D006801:Humans; D007525:Isoelectric Focusing / Q000295:instrumentation*; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*
D001798:Blood Proteins; D011506:Proteins
Free-flow electrophoresis; Preparative isoelectric focusing; Protein fractionation; pH gradient