Western Blotting
免疫印迹是一种检测、分析和定量蛋白质的成熟分析技术，已被广泛用于在组织匀浆和细胞裂解液等复杂样品中检测特定的蛋白质分子。该技术通常会涉及通过凝胶电泳进行蛋白分离，然后将其转印到聚偏二氟乙烯（PVDF）或硝酸纤维素膜上。完成蛋白转印后，可对其进行染色观察并通过N端测序、质谱或免疫检测进行直接鉴定。
在免疫印迹检测中，通过蛋白质与特异性抗体的结合来鉴定蛋白质。通常，在使用适当底物进行的化学发光或比色检测中，会联合使用一抗与偶联了HRP或AP的二抗。此外，也可使用荧光标记的一抗或二抗进行直接观察。
免疫印迹已被广泛用于在生物化学领域检测特定蛋白的存在、确定翻译后修饰的程度、验证克隆应用中的蛋白表达、分析蛋白和生物标志物的表达水平、用于抗体表位定位以及检测临床环境中的疾病标志物。
随着对使用有限样品同时分析更多蛋白的需求不断提升，现有研究开始倾向于提升印迹技术的灵敏度和速度。例如，双重印迹可消除由非特异性相互作用引起的假阳性，Far-Western blotting可实现特异性蛋白间相互作用的检测，Southwestern blotting可用于鉴定与特异性DNA序列相互作用的蛋白，多次剥离印迹可提高通量并将印迹间的变异性降至最低。同时，其他新技术也在不断开发，以期减少产生信号所需的蛋白量并提高免疫印迹的定量能力。
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相关实验方案
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工作流程
凝胶电泳
Protein gel electrophoresis is used to separate and resolve proteins prior to blotting. The prepared protein mixture is run on a polyacrylamide gel to sort proteins by molecular weight and charge.
转印到膜
The proteins separated on the gel by electrophoresis are immobilized by transfer onto a PVDF or nitrocellulose membrane. The transfer uses electric current to pull proteins from the gel onto the membrane (electroblotting).
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