Dong Ming, Choi Megan, Biggin Mark D, Jin Jian
Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
Methods Mol Biol. 2012;869:373-84. doi: 10.1007/978-1-61779-821-4_30.
We developed a multichannel gel electrophoresis system that continuously collects fractions as protein bands migrate off the bottom of gel columns. The device uses 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 allows continuous and simultaneous fraction collection from multiple channels at low cost. Using the system with SDS-PAGE, 300 μg samples of protein can be separated and eluted into 48-96 fractions over a mass range of 10-150 kDa in 2.5 h. Each eluted protein can be recovered at 50% efficiency or higher in ∼500 μL. The system can also be used for native gel electrophoresis, but protein aggregation limits the loading capacity to about 50 μg per channel and reduces resolution. This system has the potential to be coupled with mass spectrometry to achieve high-throughput protein identification.
我们开发了一种多通道凝胶电泳系统，该系统可在蛋白质条带从凝胶柱底部迁移出来时持续收集馏分。该装置使用几根短的线性凝胶柱，每根凝胶柱的丙烯酰胺百分比不同，以实现与长梯度凝胶相似的分离能力。“逆流自由流动”洗脱技术能够以低成本从多个通道连续且同时收集馏分。使用该系统进行SDS - PAGE时，在2.5小时内可将300μg蛋白质样品分离并洗脱成48 - 96个馏分，质量范围为10 - 150 kDa。每个洗脱的蛋白质可在约500μL中以50%或更高的效率回收。该系统也可用于天然凝胶电泳，但蛋白质聚集会将每个通道的上样量限制在约50μg，并降低分辨率。该系统有潜力与质谱联用，以实现高通量蛋白质鉴定。
