Van Dam A P, Van den Brink H G, Smeenk R J
Central Laboratory of the Red Cross Blood Transfusion Service, Amsterdam, The Netherlands.
J Immunol Methods. 1990 May 8;129(1):63-70. doi: 10.1016/0022-1759(90)90421-q.
When the immunoblotting technique is used as a diagnostic tool, the reproducibility of the results is a major problem. When purified radiolabelled proteins were applied onto SDS gels, the recovery of radioactivity on the blot after electrophoresis, blotting and incubation ranged from 10 to 65%, depending on the protein. Although the addition of SDS was subsequently shown to improve protein transfer from gel to blot, it is not recommended because immunological recognition of proteins is diminished after this transfer step. We suggest that during the incubation of protein blots detergents are necessary not only to diminish non-specific background, but also to renature proteins. However, since these detergents also elute protein from nitrocellulose and other blotting matrices, they are in part responsible for the lack of reproducibility in immunoblotting results.
当免疫印迹技术用作诊断工具时，结果的可重复性是一个主要问题。当将纯化的放射性标记蛋白质应用于SDS凝胶时，根据蛋白质的不同，电泳、印迹和孵育后印迹上的放射性回收率在10%至65%之间。尽管随后发现添加SDS可改善蛋白质从凝胶到印迹的转移，但不建议这样做，因为在此转移步骤后蛋白质的免疫识别会减弱。我们建议在蛋白质印迹孵育过程中，去污剂不仅对于减少非特异性背景是必要的，而且对于使蛋白质复性也是必要的。然而，由于这些去污剂也会从硝酸纤维素和其他印迹基质上洗脱蛋白质，它们在一定程度上导致了免疫印迹结果缺乏可重复性。
