Choosing A Primary Antibody Or Secondary Antibody

The nature of your sample will determine which antibody is most appropriate. Consider the following aspects:

The region of the protein that you wish to detect

Antibodies are produced by immunizing the host animal with an immunogenic substance. Immunogens can be whole proteins, protein fragments, peptides, whole organisms (eg bacteria), or cells. You can choose the antibodies through reliable antibody production companies via

Immunogens are usually described in datasheets (in some cases, for their own reasons, no detailed description of immunogens is provided).

Check if the immunogen is identical to or in the region of the protein you are trying to find. For example, if you are trying to detect cell surface proteins in living cells with FACS, select antibodies directed against the extracellular domain of the protein.

The difference between primary antibody and secondary antibody - Cusabio

Sample processing

Some antibodies require special sample handling. Many antibodies only recognize reduced and denatured proteins because this represents an epitope that would otherwise be obscured by secondary and tertiary folding of the protein.

On the other hand, some antibodies only recognize the epitope on the protein in its natural and folded state. Our western blotting antibodies require sample reduction and denaturation unless otherwise specified in the data sheet.

Choice of primary host antibody type

The species in which the primary antibody is raised must be different from the species in your sample. This is done to avoid cross-reactivity between secondary anti-immunoglobulin antibodies and endogenous immunoglobulins in the sample.

For example, if you are studying mouse protein, select the primary antibody that is generated in a different form from the mouse. Rabbit-bred primary antibody would be the right choice, followed by rabbit-bred secondary IgG antibody.

Secondary antibody choice

The secondary antibody must be directed against the host type of the primary antibody you are using. For example, if your primary mouse is monoclonal, you will need a secondary anti-mouse. Check the secondary antibody datasheet to make sure it’s been tested in the app you’re using.