How to choose a secondary antibody?
For secondary antibodies, besides the common question about difference between primary antibody and secondary antibody, how to select a right secondary antibody for your experiment is the ultimate question. If you want to solve this question, you need to consider the following aspects:
- The host species of primary antibody
This is the first step in our search for a suitable secondary antibody. It should also be the principle that everyone is most familiar with: which species the primary antibody should come from, and which species the secondary antibody should resist.
- Class or subclass of primary antibody:
The light chain of different types of antibodies can be divided into kappa (κ) type or lambda (λ) type; and the structure of the heavy chain determines the class of each antibody. Strictly speaking, the secondary antibody needs to match the same type of primary antibody. About this point, you can view the question named what is the class (isotype) and/or subclass of the primary antibody to acquire more information.
- Secondary antibody specificity
Specific for the entire antibody molecule (H+L): such as anti-IgG (H+L), this kind of antibodies can bind to various structural components of the antibody and can react with other immunoglobulins (IgM or IgA). Because all immunoglobulins have the same light chain (κ or λ).
Specific for Fab fragments: this kind of antibodies can bind to both heavy and light chains, and since they can react with light chains, they can also bind to other immunoglobulins with the same light chain.
Specific for Fc fragments or heavy chains: These antibodies react with the Fc region of the heavy chain and, therefore, they are class specific. For example, a γ-chain-specific antibody reacts only with IgG, and a μ-chain-specific antibody reacts only with IgM.
Specific for light chains: reacts with all classes of antibodies.
- Coupling label for secondary antibody
In general, secondary antibodies can be either enzyme labeled (horseradish peroxidase HRP and alkaline phosphatase AP or its derivatives APAAP, PAP), fluorescence labeled (FITC, Alexa-Fluor, Qdot) or biotin conjugated. The secondary antibody to which label is selected depends primarily on the specific experiment.