How to find the antibodies most suitable for your experiments.
There is often more than one antibody available for a target. To narrow your choice, you need to consider several aspects of your experiment:
Antibody datasheets list the applications we have tested and found the antibody to work in. If an application is not listed, it means that we have not tested it and it is unknown how the antibody will perform.
When an antibody is tested in an application and failed, this is noted on the datasheet. Our antibodies are continuously tested in-house and datasheets are updated with latest application information.
For assistance with your application, browse our protocol library.
The nature of the sample determines which antibody is most appropriate. Consider the following aspects:
Choose an antibody that has been raised against the same species your sample is from. The antibody may react with the same target protein from other species sharing sufficient amino acid sequence homology.
If your sample is not from one of the species listed in the datasheet, this means that the species has not been tested and we cannot demonstrate suitability. A prediction of cross-reactivity is made based on sequence similarity.
The species the primary antibody is raised in should be different from the species of your sample. This is to avoid cross-reactivity of the secondary anti-immunoglobulin antibody with endogenous immunoglobulins in the sample.
For instance, if you are studying a mouse protein, choose a primary antibody that is raised in a different species. A primary antibody raised in rabbit will be an appropriate choice, followed by an anti-rabbit IgG secondary antibody conjugated to a detection molecule (enzyme, fluorochrome, biotin, etc.). This issue can be avoided if a conjugated primary antibody is used.
For techniques using samples that do not contain endogenous immunoglobulin (IgG), the choice of host species of the primary antibody is less critical. An example is western blotting of a cell lysate that is not expected to contain IgG. However, tissue lysates and tissue culture supernatants that contain serum will contain immunoglobulins. IgG will appear in western blots of reduced, denatured samples as bands at 50 and 25 kDa corresponding to the heavy and light chains of the IgG molecule.
Secondary antibodies should be against the host species of the primary antibody you are using. For example, if your primary is a mouse monoclonal, you will require an anti-mouse secondary.
Check the datasheet of the secondary antibody to ensure it is tested in the application you will be using. Browse our range of secondary antibodies conjugated to a variety of fluorochromes and chromogens and view our more detailed page on How to choose a secondary antibody.
Double immunostaining of cell cultures or tissue sections using conjugated primary antibodies requires these antibodies to be raised in different species and that the secondary antibodies recognize one of the species exclusively.
Choose from our range of secondary anti-Ig antibodies, which have been pre-adsorbed against immunoglobulins from other species to remove cross-reactivity.
Labels are conjugated (joined) to antibodies in order to visualize the binding of the antibody. The choice of label depends on the experimental application. Read our useful guide on how to choose the correct conjugated secondary antibody.
Fluorescent labels emit light in the visual range when excited by light of a specific wavelength. There are several available, all with their own excitation and emission characteristics.
Biotinylated antibodies are useful for amplification of signal when followed by an avidin-biotin-enzyme or fluorochrome complex (commonly abbreviated as "ABC" reagent), or avidin or streptavidin conjugated to an enzyme or fluorochrome.