title: Target protein separation and preparation by free-flow electrophoresis coupled with charge-to-mass ratio analysis.
abstract: Herein, a novel strategy was developed to separate and prepare target protein from complex sample by free-flow electrophoresis (FFE), which mainly based on the charge-to-mass ratio (C/M) analysis of proteins. The C/M values of three model proteins, namely Cytochrome C (Cyt C), myoglobin (Mb) and bovine serum albumin (BSA) were analyzed under different pH and the separation of these proteins was predicted by CLC Protein Workbench software. Series of experiments were performed to validate the proposed method. The obtained data showed high accordance with our prediction. In addition, the chamber buffer (CB) of FFE system was optimized to improve the resolution of separation. Meanwhile, in order to evaluate the analytical performance of the proposed method, Cyt C was extracted from swine heart and further separated by FFE based on C/M analysis. Results showed that Cyt C was completely separated from the crude sample and a purity of 96.9% was achieved. The activity of prepared Cyt C was 98.3%, which indicate that the proposed method is promising in a wide variety of research areas where the native properties of proteins should be maintained for downstream analysis.
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Qiao-Yi
authors.initials: QY
authors.lastname: Shen
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Chen-Gang
authors.initials: CG
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; Institute of Refrigeration and Cryogenics, School of Mechanical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Yan
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Hai-Yang
authors.initials: HY
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Sharmin
authors.initials: S
authors.lastname: Jahan
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Liu-Yin
authors.initials: LY
authors.lastname: Fan
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: huaxiao@sjtu.edu.cn.
authors.forename: Hua
authors.initials: H
authors.lastname: Xiao
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: cxcao@sjtu.edu.cn.
authors.forename: Cheng-Xi
authors.initials: CX
authors.lastname: Cao
mesh_terms: D000818:Animals; D002021:Buffers; D002623:Chemistry Techniques, Analytical / Q000379:methods* / Q000592:standards; D045304:Cytochromes c / Q000032:analysis / Q000737:chemistry; D004586:Electrophoresis; D009206:Myocardium / Q000737:chemistry; D009211:Myoglobin / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry; D013552:Swine
chemical_list: D002021:Buffers; D009211:Myoglobin; D011506:Proteins; D012710:Serum Albumin, Bovine; D045304:Cytochromes c
keywords: Cytochrome C; Preparative electrophoresis; Protein purification; Separation optimization