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Key Considerations in Choosing Antibodies

Antibody choice must consider the application, target, species, and validation methods for the best possible chance of success and reproducibility

by
Brandoch Cook, PhD

Brandoch Cook, PhD, is a freelance scientific writer. He can be reached at: brandoch.cook@gmail.com.

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Antibodies are some of the most powerful tools in biomedical science. The ability to single out one antigen of one particular protein in a species- and context-specific manner from the hundreds of thousands known, and seemingly infinite number possible, is profound. However, the options available can be bewildering and fraught with complications, leading to scientific dead ends.

So, how does one choose among millions of commercially available antibodies? Antibody-based detection serves many platforms, from biochemical and cell biological characterizations of protein expression and interaction, to companion diagnostic technologies that assess tissue disease markers. A stringent approach to antibody selection can (1) leverage the power of the chosen application to achieve sound results, and (2) avoid pitfalls inherent in the variety of hosts, clonalities, preparations, and validation parameters that contribute to the reproducibility crisis facing biomedical science.

When choosing antibodies, the investigator should address, at a minimum, the following questions:

What is the application?

An application benefits from, and often requires, a specific antibody variant or preparation. Common antibody-based applications include western blotting, ELISA, immunofluorescence, flow cytometry, immunohistochemistry (IHC), and immunoprecipitation.

An antibody that only recognizes a target antigen on a denatured protein is potentially suitable for western blotting to assess overall expression in that cell type. Alternatively, an antibody that recognizes an antigen in its native state is more suited to immunofluorescence and assessment of subcellular localization.

It is almost always better to use an antibody that has been extensively validated for your application, rather than a general-purpose antibody...

Some IHC antibodies are only optimized for unfixed frozen tissue, while others are compatible only with fixed paraffin-embedded tissue and require an additional antigen retrieval step to reveal the relevant immunogen.

Is the antibody validated for the application of interest?

It is almost always better to use an antibody that has been extensively validated for your application rather than a general-purpose antibody with reported success in other applications. Commercial antibody specification sheets should indicate, with evidence in the form of high-quality images and suggested dilution ranges, for which applications their product has been validated. Suppliers should also cite recent literature in which the product has been used. This is usually a valuable resource for the investigator to formulate specific protocols and serves as additional external validation. 

In the absence of thorough validation, the user may have to perform additional complex validation steps, including key controls, to satisfy peer-review guidelines when optimizing an application and preparing a study for publication.

Antibodies for applications with high validation standards (such as chromatin immunoprecipitation) can often work in applications with lower standards (such as western blotting), but not the other way around.

What is the exact target of interest?

Individual proteins often have multiple confounding names, sometimes with many isoforms and post-translational modifications. Databases such as UniProt can narrow searches appropriately. Moreover, knowing the biology of your protein (Is it a nuclear transcription factor? A cell-membrane receptor? A kinase that is actively phosphorylated and de-phosphorylated?) is crucial to finding the right product.

You must also know how the antibody was raised. Was it raised against a specific epitope, or against the whole protein? If the former, is the epitope available to be recognized in your application? If the latter, is the antibody cross-specific for other isoforms? Are you trying to detect a rare endogenous protein, a ubiquitous and highly expressed protein, or a tagged and exogenously expressed construct?

What species and sample type are being investigated?

If you are studying a human or mouse protein, there is often at least one validated commercial antibody available. However, in other standard research model organisms such as chick, zebrafish, and Xenopus, there is a comparatively poor menu of antibodies. Cross-specific detection is sometimes possible, but it is always dependent on the degree of interspecific homology at the epitope against which the antibody was raised.

What host species and antibody type are optimal?

The species in which an antibody was raised should be different from the species used to generate your biological sample to avoid cross-reaction with endogenous immunoglobulin molecules. Hence, one usually sees label qualifiers such as “r α m,” meaning a rabbit anti-mouse antibody.

Moreover, knowing the biology of your protein...is crucial to finding the right product.

Polyclonal antibodies, often raised in rabbits, are usually lower-cost options that give strong, reproducible signals, but may be prone to non-specific background or multiple confounding bands. Monoclonal antibodies, usually raised in mice, are homogeneous preparations recognizing a single epitope and are therefore highly specific, but they may vary from lot to lot. Recombinant monoclonals raised in host cell cultures provide the highest degree of specificity via precise definition of their DNA sequences. 

What detection method is being used?

Antibody-based applications usually proceed by detection of a chromogenic, fluorescent, or luminescent signal generated by (1) binding of the protein-specific primary antibody to a host species-matched general secondary antibody, and (2) activation of the material conjugated to the secondary antibody (usually an enzyme that acts upon a substrate, or a fluorophore that emits light).

Primary antibodies conjugated to fluorophores are commonly used for direct detection in flow cytometry. However, in most applications, conjugated secondaries are more sensitive, versatile, and cost-effective.

How suitable is the antibody to combinations with others for multiplexing?

In flow cytometry, direct co-detection of multiple proteins with conjugated primary antibodies is common. However, the emission spectra of different fluorophores may overlap or extend into different channels beyond the capability to correct signals via compensation.

For secondary antibody-based multiplexing in other applications, antibodies raised in different species must be used, and should exclusively recognize the designated species. 

Answering these questions to formulate a firm plan is the first step in purchasing antibodies with the best chance to obtain reliable results. Understanding a target protein’s biology, knowing the sample type and species, and assessing the degree of validation within your application of interest will inevitably lead in the right direction. But when in doubt, ask for a trial size.