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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:
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.
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.
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.
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Review : "This is an excellent antibody to endoplasmin in HC11 cells. Clean, reliable detection with very little if any background. Great antibody for Westerns. Could probably be optimized for 1 hour primary incubation."
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