探讨了电转移中蛋白质的透膜现象及其对蛋白质印迹结果的影响.采用抗凋亡抑制蛋白-Survivin的抗体,对细胞裂解液进行蛋白质印迹.与常规操作方法不同之处是:在电转移的凝胶“三明治”中,重叠放置两张硝酸纤维素膜.电转移后,对两张膜同时进行免疫印迹.在特定的转移条件下,两张膜的免疫印迹都出现了Survivin蛋白的特异印迹带,证实了电转移中存在着蛋白质的透膜现象.转移时间、电流强度和蛋白质的分子质量,都是影响蛋白质透膜的相关因素.电转移中蛋白质的透膜,可以产生“印迹复制失真”的效应,从而最终影响蛋白质印迹定性和定量的结果.所得实验结果和结论,揭示了蛋白质印迹技术方法学中一个需要加以充分关注的问题,对于科学掌握和应用该技术具有积极作用.
To elucidate the phenomenon of protein blowthrough in electrophoretic transfer and its effects on Western blotting. Using survivin antibody (a novel inhibitor of apoptosis protein), several kinds of cancer cell lysate were analyzed by Western blotting. Comparing with the traditional transfer method, two pieces of nitrocellular membranes were applied in the transfer “sandwich”. Then these two membranes were analyzed by Western blotting under same conditions. In some electrophoretic transfer circumstances, the specific Western blotting band corresponding to survivin were detected in both of them. This revealed the phenomenon of protein blowthrough in electrophoretic transfer. The length of transfer time, electric current and the molecular mass of targeted protein all affected the effects of protein blowthrough. Protein blowthrough will lead to “blotting distortion” and affect the result of quantitative analysis and qualitative analysis in Western blotting. The result revealed an important problem which required to pay more attention to in Western blotting. And it will make some help for scientifically mastering and using this technique.
董燕,张枫,梅柱中,刘斌,孙志贤.电转移中蛋白质的透膜现象及其对蛋白质印迹结果的影响[J].生物化学与生物物理进展,2002,29(3):449-453
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