title: Novel moving reaction boundary-induced stacking and separation of human hemoglobins in slab polyacrylamide gel electrophoresis.
abstract: We developed a novel polyacrylamide gel electrophoresis (PAGE) method to stack and separate human hemoglobins (Hbs) based on the concept of moving reaction boundary (MRB). This differs from the classic isotachophoresis (ITP)-based stacking PAGE in the aspect of buffer composition, including the electrode buffer (pH 8.62 Tris-Gly), sample buffer (pH 6.78 Tris-Gly), and separation buffer (pH 8.52 Tris-Gly). In the MRB-PAGE system, a transient MRB was formed between alkaline electrode buffer and acidic sample buffer, being designed to move toward the anode. Hbs carried partial positive charges in the sample buffer due to its pH below pI values of Hbs, resulting in electromigrating to the cathode. Hbs would carry negative charges quickly when migrated into the alkaline electrode buffer and be transported to the anode until meeting the sample buffer again. Thus, Hbs were stacked within a MRB until the transient MRB reached the separation buffer and then separated by zone electrophoresis with molecular sieve effect of the gel. The experimental results demonstrated that there were three clear and sharp protein zones of Hbs (HbA1c, HbA0, and HbA2) in MRB-PAGE, in contrast to only one protein zone (HbA0) in ITP-PAGE for large-volume loading (≥15 μl), indicating high stacking efficiency, separation resolution, and good sensitivity of MRB-PAGE. In addition, MRB-PAGE was performed in a conventional slab PAGE device, requiring no special device. Thus, it could be widely used in separation and analysis of diluted protein in a standard laboratory.
authors.affiliation: Laboratory of Analytical Biochemistry and Bioseparation, State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Rd, Shanghai, 200240, China.
authors.forename: Yun-Yun
authors.initials: YY
authors.lastname: Tang
authors.forename: Hou-Yu
authors.initials: HY
authors.lastname: Wang
authors.forename: Lu
authors.initials: L
authors.lastname: Chen
authors.forename: Si
authors.initials: S
authors.lastname: Li
authors.forename: Chen-Gang
authors.initials: CG
authors.lastname: Guo
authors.forename: Hui-Zhi
authors.initials: HZ
authors.lastname: Fan
authors.forename: Cheng-Xi
authors.initials: CX
authors.lastname: Cao
authors.forename: Liu-Yin
authors.initials: LY
authors.lastname: Fan
mesh_terms: D002021:Buffers; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D006454:Hemoglobins / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration
chemical_list: D002021:Buffers; D006454:Hemoglobins