Choosing secondary antibodies for immunohistochemistry - By: Arthor Greenwald

Conjugated and non-conjugated secondary antibodies are routinely used to detect proteins in immunoassays. Antibody databases are becoming ever more expansive, which can be confusing. Here, we give a brief overview of how to choose the best products for your application.

Whereas primary antibodies (1o Ab's) target a specific antigen, secondary antibodies (2o Ab's) target specific areas of the primary proteins. They must be raised against the same species as the 1o Ab (mouse to mouse etc) It is also important secondary Abs match their primary equivalent. For example, if a1o Ab is rabbit IgM, the secondary protein should be anti-rabbit IgM.

Monoclonal IgG proteins have subclasses - IgG1, IgG2 a - c, IgG3 etc. Sometimes, these classes are specified, but often they are unknown. However, anti-mouse IgG recognises most of the subclasses, so is sufficient for most applications. Occasionally, you may want a specific subclass to subclass match if, for example you are using a double-labelling technique. Suppliers like us at Novus Biologicals have a huge range of primary and secondary proteins on our databases, meaning if a monoclonal primary Ab is murine IgG2a, for example, we will probably have a secondary anti-mouse IgG2a to complement it.

Typically, affinity purified immunoglobulins are used in research. Serum is first raised to a specific antigen. The same peptide is then conjugated to an inert substrate - typically agarose beads - which are then exposed to the serum. Serum antibodies specific for that peptide bind to the beads and are then collected. The alternative is to use the raw serum. However, this is not recommended in immunoassays.

Since antibodies are antigen-specific, and raised in SPF (specific pathogen free) animals, you may ask why? Well, immunohistochemistry demands very specific signals, and Abs must be highly specific to the peptide sequence in question. Animals are constantly producing immunoglobulins to different proteins, and so non-affinity purified samples will give noisy, non-specific binding signals.

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