Institute of Plant Nutrition and Soil Science, Christian Albrechts University, Hermann-Rodewald-Str. 2, 24118 Kiel, Germany.
J Immunol Methods. 2010 Oct 31;362(1-2):89-94. doi: 10.1016/j.jim.2010.09.006. Epub 2010 Sep 15.
Western transfer after the electrophoretic separation of proteins onto an adsorbent membrane, with subsequent immunodetection, is a powerful tool for detecting and characterizing a multitude of proteins. An important aspect of the study of proteins is that they often exist as isoforms with structural microheterogeneity giving rise to differences in biological activity. Western blotting (WB) in combination with two-dimensional SDS-polyacrylamide gel electrophoresis (2D-SDS-PAGE) allows the specific quantification of single isoforms of a protein. We have investigated whether a methodical modification of 2D-SDS-PAGE improves the quality of quantifiable 2D-WB data. The effect of a combined separation of three previously electrofocused protein extracts lying side by side on a single SDS-gel in parallel has been tested against the traditional procedure, viz., the separation of one protein extract per SDS-gel. The modified procedure results in a more reliable and better quality data than the traditional procedure, which seems to be prone to producing systematic or random errors. Our simple practical procedure improves immunoblotting accuracy by excluding numerous sources of errors and saves time, immunoblotting reagents and costly antibodies.
蛋白质在吸附膜上经电泳分离后转移到Western 印迹上，然后进行免疫检测，这是一种检测和鉴定多种蛋白质的有效工具。蛋白质研究的一个重要方面是，它们通常以具有结构微异质性的同工型形式存在，从而导致生物活性的差异。Western 印迹（WB）与二维 SDS-聚丙烯酰胺凝胶电泳（2D-SDS-PAGE）相结合，可以对蛋白质的单个同工型进行特异性定量。我们研究了对 2D-SDS-PAGE 进行系统的改进是否能提高可定量 2D-WB 数据的质量。我们已经测试了将三个先前并排进行等电聚焦的蛋白质提取物在单个 SDS-凝胶上进行平行组合分离的效果，与传统方法（即每个 SDS-凝胶分离一个蛋白质提取物）进行对比。与传统方法相比，改进后的方法产生的数据更可靠，质量更好，传统方法似乎容易产生系统或随机误差。我们的简单实用程序通过排除许多误差源来提高免疫印迹的准确性，并节省时间、免疫印迹试剂和昂贵的抗体。
