Secondary antibodies are available in whole antibody and antibody fragment formats. Determining whether to use a whole antibody or a fragment of antibody is often application-dependent. For instance, antibody fragments are best for immunohistochemistry and immunofluorescence because of their smaller size and ability to penetrate tissues. Whole antibodies are the most frequently used secondary antibodies in most other applications.
Whole antibodies, containing heavy and light chains, provide strong divalent binding to the variable regions while providing a sufficient constant region for attachment of signal-generating dyes or enzymes. However, whole antibodies can result in high background and lower specificity because the light chain is shared by all immunoglobulins and increases cross-reactivity.
F(ab’)2 fragments result from digestion of a whole antibody with pepsin. These antibody fragments contain the strong divalent binding found in the variable regions but lack the Fc portion of the antibody. Therefore, F(ab’)2 fragments are often the preferred type of secondary antibody for use with samples where Fc receptors may be present. They are also smaller than whole antibodies and may penetrate some samples better. However, the smaller size also means that fewer dyes or enzymes can be conjugated to the F(ab’)2 fragment, resulting in secondary antibodies that may be less sensitive than whole antibody options. Fab’ fragments result from digestion of a whole antibody with papain. These antibody fragments contain a single binding domain, with a small portion of constant region. Fab’ fragments may be used in specific applications where the bivalent antibody may be less advantageous.
Figure 3.Names and structures of antibody fragments.
If you intend to perform indirect detection with secondary antibodies, you should ideally choose a primary antibody raised in a different species to your sample. This allows you to avoid cross-reactivity of the secondary (anti-immunoglobulin) antibody with endogenous immunoglobulins in the sample.
If you intend to perform indirect detection with secondary antibodies, you should ideally choose a primary antibody raised in a different species to your sample. This allows you to avoid cross-reactivity of the secondary (anti-immunoglobulin) antibody with endogenous immunoglobulins in the sample.
A good starting concentration for a typical secondary antibody in that concentration range would be a dilution of 1:1,000. If you find your staining to be extremely bright, or that you have too much background, you can always try a higher dilution (from 1:2,000 to 1:10,000).
The specificity of a secondary antibody depends on the antibody sample that is injected as the immunogen and on the type of purification used to prepare the final product. As an example, consider a goat that is immunized with whole mouse IgG (all subclasses).
Choose antibodies designated specifically for western blotting or that list western blotting as an application. In addition, it is important to confirm that the antibody is specific towards the native or denatured protein, to determine if SDS-PAGE or native PAGE should be performed.
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