What information to look for before purchasing an antibody?

To secure the success of experiments with antibodies, information provided about a given catalog antibody should contain the following:

Host
The host, in which an antibody of interest was produced, is crucial information for choosing the correct secondary antibody.

Clonality
Antibodies can be polyclonal, monoclonal or recombinant. Each of these antibody formats offer advantages and disadvantages.

Antigen
A full-length protein (purified from tissue or recombinant) or a short peptide can be used to elicit a given antibody, and determines to which proteins the antibody is going to bind. The process of antigen design is shortly described here.

Species Reactivity
Antibody reactivity to a protein from a given species should be confirmed experimentally, buth can also be predicted based on sequence conservation.

Applications
Each application requires specific validation, and the product information sheet should provide information regarding which applications the antibody has been tested in. Recommended dilution provides further guidance for antibody use.

Application examples/Validation data
This section should contain detailed protocols describing how a given antibody was used. In case of Western blot technique, it should provide information about extraction buffer, protein load/well, mebrane transfer type and conditions, incubation buffers, blocking buffer, as well as primary and secondary antibody dilution.
In case of immunolocalization, the information should contain at least the type of tissue, fixation, blocking and primary and secondary antibody diltions.

References
For newly released antibodies, it may take 1-2 years before any publications are available.

The antibody validation process is explained here.

Things to consider:

Host
The most popular host for a polyclonal antibodies is rabbit. Antibodies produced in different hosts, offer various advantages.

Clonality
Polyclonal antibodies are mixtures of antibodies recognizing various epitopes on the target (from 3 to 15 amino acid long).
Monoclonal and recombinant antibody binds to one epitope only.

Antigen
If a full-length recombinant protein was used to produce an antibody to a protein which comes from a family with many isoforms, an antibody may react to all isoforms. Therefore, antibodies to short peptides are called monospecific, as they target a unique sequence within a target protein, chosen based on indepth bioinformatic analysis. If short peptides are used for antibody production, one has to ask the antibody supplier about the specific peptide location to make sure that the correct antibody used on samples from the species of interest.

Species reactivity
Antibodies should not be used without checking that the given sequence used in its production is also found in the sequence which is aimed to be detected. This is often overlooked and leads to the purchase and use of antibodies that do not match the target protein.

Applications
Antibody titer in ELISA is not representative for antibody reactivity on endogenous samples and cannot be used as any guarantee.

Application examples/Validation data
Provided examples of antibody use should also contain all detailes of the applied protocol. If an antibody detects a recombinant protein, it is not a proof of its reactivity on endogenous samples, which has to be determined in a separate experiment.

References
As there is a lack of common guidelines for how antibodies should be cited in scientific publications, more than a half of all antibodies used in research cannot be properly identified. Please make sure to always include the antibody name, product number and supplier when citing antibodies.

What information to look for before purchasing an antibody?

Technical blog

Latest

Archive