【求助】wb实验中为什么要用到二抗,一抗反应完了直接后面的步骤不行吗?我是新手,请指点^_^

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2021-08-12

问题描述：

如果我们将一抗用酶来标记,然后直接和底物反应,理论上也是行的通的,但是为什么还要用到二抗来反应呢?刚接触这个东西,请大家指点,谢谢.

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mdr

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直接用一抗和底物反应,是direct detection method,如果用二抗,则是indirect detection methodn . In the direct detection method, labeled primary antibody bindsto antigen on the membrane and reacts with substrate,creating a detectablesignal. In the indirect detection method, unlabeled primary antibody bindsto the antigen. Then, a labeledsecondary antibody binds to the primaryantibody and reacts with the substrate. 下面是他们各自的优缺点1.direct detection method [ Advantages:]1. Quick methodology since only one antibody is used. 2.Cross-reactivity of secondary antibody is eliminated. 3.Double staining is easily achieved using different labels on primary antibodies from the same host[Disadvantages :]1.Immunoreactivity of the primary antibody may be reduced as a result of labeling. 2.Labeling of every primary antibody is time consuming and expensive. 3.No flexibility in choice of primary antibody label from one experiment to another. 4.Little signal amplification2.indirect detection method: [ Advantages:] 1.Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. 2.A wide variety of labeled secondary antibodies are available commercially. 3.Since many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection, it is versatile. 4.Immunoreactivity of the primary antibody is not affected by labeling. 5.Different visualization markers can be used with the same primary antibody[Disadvantages :]1.Cross-reactivity may occur with the secondary antibody, resulting in nonspecific staining. 2.An extra incubation step is required in the procedure. direct detection method is not widely used as most researchers prefer the indirect detection method for a variety of reasons.

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cameron

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楼上的说得相当清楚了.事实上,二抗的引入就重要的我认为还是标记的问题.如果每个一抗都要标记就很麻烦了.当然楼上所说的各自的优缺点都很详尽了.

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jiangid4

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一抗每个都要标记很麻烦，二抗是结合的是一抗的FC片段，因此可以大批量的生产二抗

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