Immunoblotting.
Immunoblotting.
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免疫印迹。
DOI：
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发表时间：
2020
影响因子：
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通讯作者：
Larisa Litovchick
中科院分区：
文献类型：
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作者：
Larisa Litovchick
免疫印迹法允许检测固定在蛋白质保留膜支持物如硝酸纤维素或聚偏二氟乙烯（PVDF）上的蛋白质抗原。目标蛋白的检测依赖于特异性识别暴露在膜上的目标蛋白的抗体的结合。感兴趣的蛋白质可以被纯化或与细胞或组织提取物中的其他蛋白质混合。通常，免疫印迹法将通过凝胶电泳的蛋白质解析与免疫化学检测相结合，并且被称为“蛋白质印迹法”。免疫印迹可用于确定样品中目标蛋白的存在和稳态水平，其相对分子量以及蛋白在细胞组分之间的分布。免疫印迹可以使用针对合成肽抗原产生的抗体进行，所述合成肽抗原被修饰以模拟蛋白质的翻译后修饰，例如磷酸化和乙酰化，以在体内研究感兴趣的蛋白质中的这些修饰。当没有针对目标蛋白的抗体时，可以使用重组DNA技术，使用特异性识别与目标蛋白融合的重组表位标签（血凝素[HA]-、Flag-、cMyc-或谷胱甘肽-S-转移酶[GST]）的抗体进行免疫印迹。免疫印迹具有多种研究、临床和法医学应用。它也是用于表征来自多克隆血清或杂交瘤上清液的不同样品的抗体的标准技术之一。
Immunoblotting allows detection of a protein antigen immobilized on the protein-retaining membrane support such as nitrocellulose or polyvinylidene fluoride (PVDF). The detection of the protein of interest relies on the binding of an antibody that specifically recognizes the protein of interest exposed on the membrane. The protein of interest can be purified or mixed with other proteins as in cell or tissue extracts. Usually immunoblotting combines the resolution of proteins by gel electrophoresis with immunochemical detection and is referred to as "western blotting." Immunoblotting can be used to determine the presence and the steady-state level of the protein of interest in the sample, its relative molecular weight, and the distribution of the protein between cellular fractions. Immunoblotting can be performed using the antibodies raised against synthetic peptide antigens modified to mimic posttranslational modifications of proteins, such as phosphorylation and acetylation, to study these modifications in the protein of interest in vivo. When antibodies against the protein of interest are not available, immunoblotting can be performed using antibodies that specifically recognize the recombinant epitope tags (hemagglutinin [HA]-, Flag-, cMyc-, or glutathione-S-transferase [GST]) fused to the protein of interest using recombinant DNA techniques. Immunoblotting has a variety of research, clinical, and forensic medicine applications. It is also one of the standard techniques for characterization of antibodies from different samples of polyclonal sera or hybridoma supernatants.