【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..._反应底物_生物分子_生化试剂_知道

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 binds
to antigen on the membrane and reacts with substrate,creating a detectable
signal.
In the indirect detection method, unlabeled primary antibody binds
to the antigen. Then, a labeledsecondary antibody binds to the primary
antibody 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 amplification

2.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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【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...