Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients
Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients
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DOI：
10.1002/1615-9861(200202)2:2
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发表时间：
2002-02-01
期刊：
影响因子：
3.4
通讯作者：
van Oostrum, J
中科院分区：
文献类型：
--
作者：
Hoving, S;Gerrits, B;van Oostrum, J
一种可重复的高分辨率蛋白质分离方法是成功的差异蛋白质组分析的基础。在目前可用的技术中，二维凝胶电泳因其在各种实验条件下的稳健性而应用最广泛。随着在第一维中引入窄范围固定化pH梯度（IPG）条带（也称为超缩放凝胶），分析深度，即可以分解的蛋白质数量大大增加。然而，在pH值高于7的碱性区域，超变焦凝胶的等电聚焦存在分辨率和再现性问题，原因尚不清楚。为了解决这些困难，我们通过关注两个重要现象：等电聚焦过程中的水输送和二硫苏糖醇（DTT）在碱性pH梯度中的迁移，优化了碱性IPG条带上半制备量蛋白质的分离。第一个问题通过在聚焦介质中加入甘油和异丙醇得到了缓解，导致pH 7以上分辨率的显著提高。在阴极引入过量的还原剂DTT，效果更好。通过这些调整和优化的IPG条带组成，在pH 6.2-8.2范围内的分离效率现在可以与广泛使用的酸性超缩放凝胶相媲美。我们进一步证明了这些修改在pH 9.5以下的有用性，尽管在该范围内仍需要进一步改进。因此，通过扩展常规超放大凝胶覆盖的范围，可以显著增加二维凝胶电泳的分析深度，强调了该方法在差异蛋白质组学中的重要性。
A reproducible high-resolution protein separation method is the basis for a successful differential proteome analysis. Of the techniques currently available, two-dimensional gel electrophoresis is most widely used, because of its robustness under various experimental conditions. With the introduction of narrow range immobilized pH gradient (IPG) strips (also referred to as ultra-zoom gels) in the first dimension, the depth of analysis, i.e. the number of proteins that can be resolved, has increased substantially. However, for poorly understood reasons isoelectric focusing on ultra-zoom gels in the alkaline region above pH 7 has suffered from problems with resolution and reproducibility. To tackle these difficulties we have optimized the separation of semipreparative amounts of proteins on alkaline IPG strips by focusing on two important phenomena: counteracting water transport during isoelectric focusing and migration of dithiothreitol (DTT) in alkaline pH gradients. The first problem was alleviated by the addition of glycerol and isopropanol to the focusing medium, leading to a significant improvement in the resolution above pH 7. Even better results were obtained by the introduction of excess of the reducing agent DTT at the cathode. With these adaptations together with an optimized composition of the IPG strip, separation efficiency in the pH 6.2-8.2 range is now comparable to the widely used acidic ultra-zoom gels. We further demonstrated the usefulness of these modifications up to pH 9.5, although further improvements are still needed in that range. Thus, by extending the range covered by conventional ultra-zoom gels, the depth of analysis of two-dimensional gel electrophoresis can be significantly increased, underlining the importance of this method in differential proteomics.