Tovey E R, Ford S A, Baldo B A
Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, NSW, Australia.
J Biochem Biophys Methods. 1987 Jan;14(1):1-17. doi: 10.1016/0165-022x(87)90002-9.
The resolution and detection of individual components in complex extracts by protein blotting have been investigated. By probing nitrocellulose transfers with monospecific and multispecific antisera, it was demonstrated that dissociating conditions were required for the maximum resolution of antigens by polyacrylamide gel electrophoresis, a conclusion reinforced by results from 2-D electrophoresis. The dissociating and reducing treatments employed, however, were both shown to be responsible for some loss of total antigenicity and included the complete loss of at least one important antigen. Assays with nitrocelluloses of different pore sizes demonstrated that both higher protein-binding capacities and higher backgrounds were associated with the use of the smallest pore size, while the sensitivity of the assay was greatest when a non-ionic detergent, and not proteins, were used for blocking. Nitrocellulose-bound proteins may be stained with amido black, India ink, toluidine blue, Ponceau S or a gold sol, but these agents do not always give identical staining patterns. While detection of components with immuno-enzyme staining methods had some advantages, problems with non-specific binding were encountered. These did not occur with affinity purified radiolabelled second antibodies, which in combination with scanning of autoradiographs allowed a quantitative approach to be adopted.
对通过蛋白质印迹法分析复杂提取物中单个成分的分辨率和检测进行了研究。通过用单特异性和多特异性抗血清探测硝酸纤维素转移膜，结果表明聚丙烯酰胺凝胶电泳要实现抗原的最大分辨率需要解离条件，二维电泳的结果也强化了这一结论。然而，所采用的解离和还原处理均被证明会导致总抗原性有所损失，其中至少一种重要抗原会完全丧失。对不同孔径硝酸纤维素的检测表明，孔径最小的硝酸纤维素具有更高的蛋白质结合能力和更高的背景，而当使用非离子去污剂而非蛋白质进行封闭时，检测的灵敏度最高。结合在硝酸纤维素上的蛋白质可用氨基黑、印度墨汁、甲苯胺蓝、丽春红S或金溶胶染色，但这些试剂并不总是给出相同的染色模式。虽然免疫酶染色法检测成分有一些优点，但存在非特异性结合问题。亲和纯化的放射性标记二抗不存在这些问题，结合放射自显影片扫描可采用定量方法。
