Kane Lesley A, Yung Christina K, Agnetti Giulio, Neverova Irina, Van Eyk Jennifer E
Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21224, USA.
Proteomics. 2006 Nov;6(21):5683-7. doi: 10.1002/pmic.200600267.
Separation of basic proteins with 2-DE presents technical challenges involving protein precipitation, load limitations, and streaking. Cardiac mitochondria are enriched in basic proteins and difficult to resolve by 2-DE. We investigated two methods, cup and paper bridge, for sample loading of this subproteome into the basic range (pH 6-11) gels. Paper bridge loading consistently produced improved resolution of both analytical and preparative protein loads. A unique benefit of this technique is that proteins retained in the paper bridge after loading basic gels can be reloaded onto lower pH gradients (pH 4-7), allowing valued samples to be analyzed on multiple pH ranges.
利用双向电泳分离碱性蛋白质存在技术挑战，包括蛋白质沉淀、上样量限制和拖尾现象。心脏线粒体富含碱性蛋白质，难以通过双向电泳进行分离。我们研究了两种将该亚蛋白质组样品加载到碱性范围（pH 6 - 11）凝胶中的方法，即杯上样法和纸桥法。纸桥加载法始终能提高分析型和制备型蛋白质上样量的分辨率。该技术的一个独特优势是，在加载碱性凝胶后保留在纸桥中的蛋白质可以重新加载到较低pH梯度（pH 4 - 7）上，从而使珍贵样品能够在多个pH范围内进行分析。
