Towbin H, Ozbey O, Zingel O
Proteomics Group, Novartis Pharma AG, Basel, Switzerland.
Electrophoresis. 2001 Jun;22(10):1887-93. doi: 10.1002/1522-2683(200106)22:10<1887::AID-ELPS1887>3.0.CO;2-Q.
Post-translational modifications such as phosphorylation and acetylation are important elements for regulating the activity of enzymes or structural proteins. These modifications give rise to isoforms that are often not resolved by separation methods relying on the size of proteins. Here, we optimized an isoelectric focusing (IEF)-immunoblotting method suitable for analyzing protein isoforms in total cell extracts. The separations were carried out in parallel on commercially available immobilized pH gradient slab gels (IPG). The buffer used for separation contained urea, thiourea, dithiothreitol, as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), and was designed to match those used in two-dimensional polyacrylamide gel electrophoresis (PAGE) separations where efficient solubilization is required. Proteins were transferred to membranes by passive diffusion in the presence of 4 M guanidinium chloride using protocols optimized for several protein classes (tubulin, stathmin, 14-3-3 proteins) some of which required removal of CHAPS prior to transfer. In conjunction with narrow-range pH gradient gels, excellent resolution of isoforms differing by phosphorylation or acetylation was achieved. The usefulness of pI and titration curve calculations for predicting the pI shifts expected for post-translational modifications of proteins with known amino acid composition was demonstrated. Using stathmin--which contains four phosphorylation sites--as an example, the effects on the pI-shifts were well predicted. This sensitive and widely applicable IEF-blotting technology is expected to be especially suited for analyzing protein isoforms first detected by two-dimensional electrophoresis.
磷酸化和乙酰化等翻译后修饰是调节酶或结构蛋白活性的重要因素。这些修饰会产生异构体，而依赖蛋白质大小的分离方法通常无法分辨这些异构体。在此，我们优化了一种等电聚焦（IEF）-免疫印迹法，适用于分析全细胞提取物中的蛋白质异构体。分离在市售的固定化pH梯度平板凝胶（IPG）上平行进行。用于分离的缓冲液含有尿素、硫脲、二硫苏糖醇以及去污剂3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐（CHAPS），其设计与二维聚丙烯酰胺凝胶电泳（PAGE）分离中所需的高效溶解所用缓冲液相匹配。蛋白质通过在4 M氯化胍存在下的被动扩散转移到膜上，使用针对几种蛋白质类别（微管蛋白、微管相关蛋白1、14-3-3蛋白）优化的方案，其中一些在转移前需要去除CHAPS。结合窄范围pH梯度凝胶，实现了对因磷酸化或乙酰化而不同的异构体的出色分辨率。证明了pI和滴定曲线计算对于预测具有已知氨基酸组成的蛋白质翻译后修饰预期的pI变化的有用性。以含有四个磷酸化位点的微管相关蛋白1为例，对pI变化的影响得到了很好的预测。这种灵敏且广泛适用的IEF印迹技术预计特别适合分析首先通过二维电泳检测到的蛋白质异构体。
