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.
Anal Bioanal Chem. 2013 Oct;405(26):8587-95. doi: 10.1007/s00216-013-7258-6. Epub 2013 Aug 4.
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.
我们开发了一种新的聚丙烯酰胺凝胶电泳 (PAGE) 方法，基于移动反应边界 (MRB) 的概念来堆叠和分离人类血红蛋白 (Hbs)。这与基于经典等电聚焦 (ITP) 的堆叠 PAGE 在缓冲液组成方面有所不同，包括电极缓冲液 (pH 8.62 Tris-Gly)、样品缓冲液 (pH 6.78 Tris-Gly) 和分离缓冲液 (pH 8.52 Tris-Gly)。在 MRB-PAGE 系统中，在碱性电极缓冲液和酸性样品缓冲液之间形成了一个瞬时 MRB，设计为向阳极移动。由于 Hbs 的 pH 值低于其等电点，Hbs 在样品缓冲液中带有部分正电荷，导致向阴极迁移。当 Hbs 迁移到碱性电极缓冲液中时，它会迅速带上负电荷，并被运送到阳极，直到再次遇到样品缓冲液。因此，Hbs 在 MRB 内被堆叠，直到瞬时 MRB 到达分离缓冲液，然后通过凝胶的分子筛效应进行区带电泳分离。实验结果表明，MRB-PAGE 中有三个清晰锐利的 Hbs 蛋白区（HbA1c、HbA0 和 HbA2），而大容量加载（≥15 μl）的 ITP-PAGE 中只有一个蛋白区（HbA0），表明 MRB-PAGE 具有高堆积效率、分离分辨率和良好的灵敏度。此外，MRB-PAGE 在常规平板 PAGE 设备中进行，无需特殊设备。因此，它可以广泛应用于标准实验室中稀释蛋白质的分离和分析。
