Tips for Antibody Validation

There are antibody validation strategies that apply to all applications and others that are application-specific.

It is critical to understand that each application must be verified independently and while validation across multiple applications can be supportive, verification of functionality and specificity in one application or model does not prove validation in another.

It is also important to consider that no single validation assay is better than another and that multiple strategies should be employed to ensure specificity and sensitivity depending on the target and application being studied.

For all applications, the following validation methods should be employed:

• Binary model: Whenever possible, antibodies should be tested in models that includes a positive and negative control. The use of a genetic or CRISPR/Cas9-based knockout, RNAi-based knockdown, endogenous cell lines or tissues with known positive/negative (or high/low) expression of the target protein is one of the best ways to verify antibody specificity. Again, this method should be applied to every application you plan to use.
• It is also important to screen your antibodies in cell/tissue panels or arrays to rule out off-target binding. Antibodies that show exquisite specificity in one model or species may have off-target binding in another model or application – it is important to validate your antibodies in the species and model you intend to use throughout your experiments.
• Recombinant proteins or transfected constructs can be used to confirm the specificity of your antibody when no other model is available, or to verify the presence or absence of cross-reactivity with homologs or isoforms. This step is especially important if you are studying a protein target that is part of a family of highly homologous proteins.
• The use of two antibodies that detect distinct antigens on the same protein is yet another way to confirm specificity. This method is most appropriate for immunoprecipitation (IP) experiments where you immunoprecipitate with one antibody and detect with the other but can be applied to other applications.  Ideally, both antibodies should detect the same band by western blot or localize to the same compartment in a cell or tissue. However, this technique should not be used in isolation since it could be possible for both antibodies to be incorrect or yield slightly different results. 

When using antibodies that detect post-translational modifications (PTMs), either site-specific or pan-reactive (e.g. phospho-tyrosine), it is critical to not only determine specificity for the target but also the site and modification of interest. In addition, it is very important to verify that that antibody will still detect the site/PTM of interest in the presence of nearby PTMs which might alter the antigen.

• Validate the antibody in cells/tissues treated with or without agonists or inhibitors that specifically modulate the PTM on the site of interest. If you are unsure of how the site is regulated, a great resource for understanding the up- and downstream PTM regulation of protein signaling is PhosphoSite Plus.
• Phospho-tyrosine, in particular, is highly immunogenic and antibodies that detect phospho-tyrosine within one context frequently cross-react with phospho-tyrosines on other proteins.  For p-Tyr-specific antibodies, it is critical to verify site and target specificity using multiple applications and validation techniques.
• Comparing the ability of the antibody to detect the PTM and site of interest in the presence of unmodified or PTM-peptide, is a useful tool to confirm specificity in any application. Application-independent methods such as peptide ELISAs (competitive and non-competitive) can also be used to validate the specificity of antibody. Similarly, peptide arrays using peptides representing all of the possible PTMs on the site of interest AND nearby sites, can be used to determine antibody specificity as well as the impact of nearby PTMs on the ability of the antibody to bind to the site of interest.
• Lastly, one can also use site-specific mutants of proteins by making positive and negative mutants to help confirm the specificity of the antibody.  For example, introducing alanine or glutamic acid in place of a phosphorylated serine or threonine can be used to understand the specificity of the antibody. These results should be interpreted with caution since any manipulation of the antigenic epitope could abrogate antibody binding. However, this can be a useful tool when no others are available (and many reviewers will ask for this experiment anyway!).

Validation of antibodies in a specific application depends on the application and the results of the general validation methods outlined above. For example, in imaging applications, ensuring that the signal localizes to the correct cell type or compartment within a cell or tissue is critical.

Using agonists or inhibitors to induce translocation, expression, degradation or other changes in relative signal can be very useful for verifying that your antibody is detecting the right target. Use the biology of the target to design experiments that will help develop a validation strategy.

For enrichment-based applications such as IP and ChIP, ensuring the IP works using two different antibodies as described above should be the first choice but one can also use heterologous, epitope-tagged proteins to validate that the antibody works in an IP assay by using the target-specific antibody for the enrichment and the tag antibody for detection. If so equipped, IP followed by mass spectrometry can also be used to verify the proteins enriched during an IP.

The importance of cross-application validation cannot be underestimated. A simple western blot prior to using an antibody for IHC can provide insight regarding the specificity and potential off-target interactions of an antibody. However, doing this correctly means using the same model for both the western blot and IHC experiments. Similarly, immunocytochemical methods can be used to confirm observations made by flow cytometry due to the similarly in methods used to fix and stain the cells.

Most critically, every experiment should include all correct positive and negative controls including experimental, loading, and isotype controls depending on the experimental design and application being used. This ensures the efficacy of the antibody but also that the experiment itself worked independent of the specificity and functionality of the antibody.