3 Guidelines for Using Secondary Antibodies -by Dr. Booster
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April 16, 2017
The purpose of using a secondary antibody is to amplify the primary antibody and antigen interactions. Before choosing a secondary antibody for your experiment, ask yourself the following 3 questions to help you choose the appropriate one:
What is the host species of the primary antibody?
The secondary antibody should be raised against the host in which the primary antibody was raised. For example, if the primary antibody was raised in rabbit, you will need an anti-rabbit secondary antibody raised in a species other than rabbit.
What is the isotype of the primary antibody?
The secondary antibody has to be directed specifically against the isotype of the primary antibody. For example, the polyclonal primary antibodies are generally of the IgG isotype and thus the corresponding (species specific) secondary antibody needs be an anti-IgG antibody. The monoclonal antibodies, which are commonly raised in mice and rabbits, have the IgG1 isotype and require the suitable secondary antibody.
Does the secondary antibody cross react with species other than that of the primary antibody?
There is always a chance that the secondary antibody might cross-react with the endogenous immunoglobulins or other proteins of the sample. Therefore, it is a good idea to go for secondary antibodies that have been pre-adsorbed with the serum of the same species as the sample.
In addition, the primary antibodies of a multicolour panel will likely have been raised in different hosts and thus the corresponding secondary antibodies might cross-react with more than one primary antibody. The solution for this is to utilize cross-adsorbed secondary antibodies which have been treated against sera of one or more species.