Knierim M, Buchholz J, Pflug W
Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg.
Anal Biochem. 1988 Jul;172(1):139-44. doi: 10.1016/0003-2697(88)90422-8.
In the present article a procedure is described which combines the horizontal isoelectric focusing (IEF) of proteins on fabric-reinforced polyacrylamide gels with the subsequent electrophoretic transfer of the proteins to nitrocellulose or Immobilon. The application of a carrier material that is permeable for current and molecules and that serves as a physical support of the IEF gel is one of the central prerequisites for the method to work. Moreover, it is important to fix the pH gradient topographically by the use of Immobilines mainly in order to avoid distortion of the protein pattern during the electrotransfer (Western blot). The Western blot can be performed either in the submerse or in the so-called "semi-dry" blotting system. Our procedure is compatible with IEF protocols employing buffer systems with or without urea. The efficacy of our method is demonstrated by the IEF and Western blotting of several known marker proteins.
在本文中，描述了一种方法，该方法将蛋白质在织物增强聚丙烯酰胺凝胶上的水平等电聚焦（IEF）与随后将蛋白质电泳转移到硝酸纤维素或Immobilon相结合。使用对电流和分子具有渗透性并作为IEF凝胶物理支撑的载体材料是该方法起作用的核心前提之一。此外，主要通过使用固定化电解质在地形上固定pH梯度很重要，以便避免在电转移（蛋白质印迹法）过程中蛋白质图谱的扭曲。蛋白质印迹法可以在浸没式或所谓的“半干”印迹系统中进行。我们的方法与采用含或不含尿素的缓冲系统的IEF方案兼容。通过对几种已知标记蛋白进行IEF和蛋白质印迹法证明了我们方法的有效性。
