CST BLOG: Lab Expectations

The official blog of Cell Signaling Technology (CST), where we discuss what to expect from your time at the bench, share tips, tricks, and information.

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You want to generate accurate results. You’ve planned out your experiment, accounting for all necessary reagents, and double-checking that you are using the proper controls. You’re excited to examine your target more closely, but have you considered the specificity of your antibody? This is a step that some researchers miss. You may have seen a validation claim from your reagent supplier, but how can you be certain that your antibody is specific?

Much has been made of knock-out (KO) validation. It can be a powerful tool to demonstrate protein expression as well as antibody sensitivity. When one knows for certain which cell lines express our target, then a knockout can be used. Unfortunately, it’s not always possible to use a knockout system. For example, many proteins are essential for a cell line to survive, and if an essential protein is knocked out, the cell line is no longer viable. Furthermore, some cell lines cannot be transfected efficiently with knockout reagents. If the target is expressed exclusively in “hard to transfect” cell lines, it is not possible to use a knockout tool for antibody validation. If a KO system is not an option for antibody validation, there are many other strategies that manufacturers can and should use to validate an antibody.


The truth is that there are many strategies that manufacturers can use to demonstrate specificity, and that a combination of validation strategies is necessary to truly validate.

We guarantee our antibodies by carefully tailoring the combination of validation strategies: binary, ranged, orthogonal, multiple antibody, recombinant, and complementary. This means customizing our validation process according to the biological role of the target, while considering the sensitivity requirements of the downstream assay, the availability of appropriate testing models, and the relevance of each method to target investigation.




A binary approach is one of the best ways to evaluate antibody specificity. By testing an antibody in biologically relevant positive and negative expression systems, it is possible to confirm that it recognizes the target antigen in its native environment without cross-reacting with other biomolecules present in the sample. Binary models include endogenous cells or tissues where expression of the target protein is known or predicted to be positive (high) or negative (low), genetic knockouts, and the use of treatments to induce or inhibit expression or modification of a protein target.


For binary testing to be effective, data should always be verified using an orthogonal method, such as genetic sequencing to confirm knockout or proteomic profiling to verify expression levels. To provide utmost confidence in antibody performance, additional validation strategies should also be employed. Moreover, each model that is used for binary validation of an antibody should be tested in each application for which the antibody is intended to be used – just because an antibody is specific by western blot does not mean that it will be as specific by immunohistochemistry (IHC). For instance, when performing a western blot the protein and epitope are denatured, whereas in IHC they remain intact, but the epitope that the antibody can bind to in IHC might be masked.




Binary models are not always readily available and can be time consuming or expensive to produce for the sole purpose of validating an antibody. Moreover, to assess the sensitivity of an antibody in the application and protocol being used, a complementary hallmark is required.


Ideally suited to this purpose, a ranged strategy includes both endogenous and heterologous models that express high, moderate, and low levels of the target of interest. A ranged approach is critical to understand the optimal working conditions of an antibody; however, its importance to the overall antibody validation process may often be overlooked. The most important (yet subtle) difference between ranged strategy and binary strategy is that ranged models rely on differences in target expression or modification that are not black and white. Typically, ranged models are more reflective of actual biology, where expression of the target is high or low in one cell line or tissue relative to another, or is modified only slightly by agonist or antagonist treatment. This means that, while ranged testing results are significant, they are not as striking or as easily interpretable as data generated by binary evaluation.




An orthogonal strategy for antibody validation involves cross-referencing antibody-based results with data obtained using non-antibody-based methods. This approach is critical to verify existing antibody validation data and to identify any effects or artifacts that are directly related to the antibody in question. In its simplest form, an orthogonal strategy dictates that results obtained using other strategies require corroboration by non-antibody-based detection methods. As just one example, positive and negative expression of the target observed by binary or ranged strategies should always be confirmed using an orthogonal approach, such as genetic sequencing to confirm knockout or transcriptomic analysis of mRNA to confirm expression.


Multiple Antibody


A multiple antibody strategy is a powerful approach to antibody validation. One of the most common methods to achieve this is to immunoprecipitate (IP) the target with one antibody and subsequently detect it by western blotting with another antibody against the same target. This provides confidence that both antibodies are binding the correct protein.


Another familiar method of multiple antibody validation involves using two or more antibodies against distinct, nonoverlapping epitopes on the same target to produce directly comparable immunostaining data. This is typically demonstrated through techniques such as western blotting, immunocytochemistry, or immunohistochemistry. By probing identical samples with multiple antibodies in parallel, it is possible to gain a relatively quick visual indication of antibody specificity.




For antigenic targets where expression of the protein is very low or unknown, the use of recombinant proteins or heterologous expression in a surrogate cell line may be necessary for antibody validation. Although endogenous systems are preferred for their closer representation of in vivo conditions, recombinant strategy offers several advantages.


Firstly, recombinant strategy can be used to verify the cross-reactivity of an antibody with protein isoforms or conserved family members, providing useful information regarding the antibody’s potential for off-target binding based on antigen homology. Recombinant strategy can also be used to test the sensitivity of an antibody through titrating the target protein by expression or dilution.




Depending on the antigenic target being studied and the application being used, it may be advisable to employ complementary strategies during antibody validation. These approaches can provide vital information regarding antibody specificity or functionality and can be carefully tailored to the biological nature of the target as well as to the exacting requirements of the downstream assay.


Complementary strategies include the use of peptide arrays and/or ELISAs to determine the specificity of an antibody for a post-translational modification (PTM), and various peptide blocking methods to prevent antibody binding to a defined antigen. Protocol optimization is also included within the complementary strategies hallmark, as are various functional assays such as neutralization or protein activation using an antibody as an agonist. All these methods provide additional data to support results generated using the other hallmarks of validation.


Evaluating Your Reagents


No one validation strategy is better than another and none of the approaches discussed here should ever be used in isolation. Each antibody-based application provides a unique set of conditions, presenting very different challenges relative to antibody specificity, sensitivity, and functionality. As just one example, an antibody that displays exquisite specificity by western blot may be nonspecific in an immunohistochemistry assay or ineffective in a functional assay. This emphasizes the importance of validating every single antibody using strategies and protocols consistent with the desired application in the intended model system. CST uses a combination of all these approaches to validate every single one of our antibodies. If you cannot determine if the antibody you are using has gone through this level of validation, you should consider validating on your own.


Learn more about antibody validation strategies.

Chris Sumner
Chris Sumner
Chris Sumner was the Editor-in-Chief of Lab Expectations. When he's not reading/writing about curing disease, he's hiking in the woods, playing guitar, or searching for the world's best lobster roll.

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