Genomics Division, Lawrence Berkeley National Laboratory, CA 94720, USA.
Electrophoresis. 2010 Jan;31(3):440-7. doi: 10.1002/elps.200900435.
To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "Counter Free-Flow" elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of approximately 10-150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 microL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 microg per channel and reduced resolution.
为了在 PAGE 蛋白质分离和质谱蛋白质鉴定之间实现直接接口，我们开发了一种多通道系统，该系统可连续收集蛋白质带从凝胶电泳柱底部迁移出的馏分。该设备使用几个短的线性凝胶柱（丙烯酰胺百分比不同）构建，以实现与长梯度凝胶相似的分离能力。然后，“Counter Free-Flow”洗脱技术可低成本地连续同时从多个通道收集馏分。我们证明，使用 SDS-PAGE 可以在该系统上实现快速，高分辨率的复杂蛋白质混合物分离。例如，在 2.5 小时的电泳运行中，每个样品在大约 10-150 kDa 的质量范围内被分离并洗脱成 48-96 个馏分；样品回收率为 50%或更高；每个通道可在 0.4 mL 中加载多达 0.3 mg 的蛋白质；纯化带可在两到三个馏分（200 μL/馏分）中洗脱。当运行天然凝胶电泳时，得到了相似的结果，但蛋白质聚集将每个通道的上样容量限制在约 50 μg，并降低了分辨率。
