Guidelines of Secondary Antibody for Western Blotting
Licia Miller Product Manager
Target species (primary antibody)
When choosing a secondary antibody for western blotting, one of the main selection criteria is the species of the primary antibody that the secondary antibody binds to. For example, if the primary antibody is a rabbit polyclonal IgG, an anti-rabbit IgG secondary antibody raised in another host species (e.g., goat, donkey, or mouse) can be used to detect the primary antibody (e.g., a goat anti-rabbit IgG secondary antibody, Ab176443).
To detect multiple specific protein targets by western blotting, it is helpful to use primary antibodies from different species. Multiplex fluorescent western blotting can then be performed using matched secondary antibodies labeled with different fluorescent dyes (e.g., Alexa Fluor Plus 488, 555, 647, or 680).
We offer a wide range of conjugated secondary antibodies suitable for fluorescent or chemiluminescent western blotting. Choose from over 15 target species to find a new secondary antibody that matches your primary antibody.
be generated to bind to the entire primary immunoglobulin, such as H+L (heavy and light chains) targeted secondary antibodies, or they can be generated to bind only to specific portions of the immunoglobulin, such as kappa vs lambda light chain specific secondary antibodies, heavy vs light chain specific secondary antibodies, or fragment specific secondary antibodies. For example, Fc fragment specific secondary antibodies, F(ab) or F(ab')2 specific secondary antibodies.
When planning a Western blot experiment, in addition to other factors such as optimizing antibody dilution, the choice of secondary antibody can also help achieve optimal results. Careful selection of secondary antibodies and optimization of dilutions can significantly improve Western blot analysis, especially when dealing with protein targets of varying abundance or high versus low affinity primary antibodies on the same Western blot membrane.
Target abundance changes are common because the loading control housekeeping protein is highly abundant in the lysate, while many protein targets of interest may be expressed at only low copy numbers. Visualizing both simultaneously on the same membrane can be challenging. The table below provides general guidelines to help leverage the unique capabilities of different secondary antibodies to improve Western blot detection.
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Host species and secondary antibody type (polyclonal, monoclonal, recombinant)
Polyclonal, monoclonal, and recombinant secondary antibodies, as well as antibody fragments, can be used in western blotting.
Polyclonal secondary antibodies are the most widely used secondary antibody format. They recognize multiple epitopes on the primary antibody and are therefore more sensitive than monoclonal antibodies that recognize only one epitope or antibody fragments without the Fc region.
The value of monoclonal secondary antibodies lies in their lot-to-lot consistency and, in many cases, extensive characterization and publication history.
Recombinant secondary antibodies have other advantages. In addition to having good properties and batch-to-batch consistency, they can be further modified at specific sites to add desired properties to native immunoglobulins, such as class switching or site-specific tagging. Recombinant antibodies can be pooled to generate recombinant antibody pools, such as recombinant polyclonal primary antibodies or superclonal recombinant secondary antibodies.
The table below shows the advantages and disadvantages of polyclonal antibodies, monoclonal antibodies, and recombinant antibodies as secondary antibodies.
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Detection method
Secondary antibodies can be conjugated to several different probes or enzymes to detect the target antigen. The choice of label depends on the application and the detection method of the secondary antibody. Enzyme reporters such as horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most commonly used in western blotting. These enzymes can be used with chemiluminescent or chromogenic detection methods. Fluorescently labeled secondary antibodies are also becoming more widely used to detect low-abundance targets with higher signal-to-noise ratios.
Cross-adsorbed antibodies
When performing multiplex western blot analysis, consider using highly cross-adsorbed secondary antibodies to limit cross-reactivity between antibodies. Cross-adsorption is a purification process that helps eliminate nonspecific antibodies from an antibody mixture, such as antibodies of a specific class, isotype, or host species.
Western Blot analysis after immunoprecipitation
When performing western blot analysis after immunoprecipitation, secondary antibody selection plays an important role. When performing immunoprecipitation, the primary antibody or control IgG is usually co-eluted with the target protein. Therefore, the eluted fractions used for western blot analysis always contain varying amounts of IgG. For example, if a rabbit polyclonal antibody is used to enrich the target protein by immunoprecipitation, the reduced and denatured heavy and light chains in the eluted fractions will be at 25 and 50 μg/ml when using a traditional HRP-conjugated anti-rabbit IgG ( H+L ) secondary antibody. A band is produced at kD because the secondary antibody binds directly to the denatured IgG.
If the heavy and light chains produced by the secondary antibody do not interfere with the target of the primary antibody, then only these bands need to be labeled as heavy and light chains. When running at kD, it may not be possible to distinguish the target band from the heavy or light chain bands.
There are several possible solutions:
• Use Clean-Blot IP detection reagent, which detects only the native primary antibody and not the denatured IgG in the immunoprecipitate elution fraction.
• If the target is ~25 kD (light chain interference), use an Fc-specific secondary antibody .
• If the target protein molecular weight is approximately 50 kD (heavy chain interference), use a light chain specific secondary antibody.
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