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CST Antibody Validation Principles

At Cell Signaling Technology (CST), we understand that there is no single assay that can determine the validity of an antibody. Confirming that an immunoreagent is sufficiently specific and sensitive depends on the application and protocol being used, the type and quality of sample being analyzed, and the inherent biophysical properties of the antibody itself.

To ensure our antibodies will work in your experiment, we adhere to the Hallmarks of Antibody Validation, six complementary strategies that can be used to determine the functionality, specificity, and sensitivity of an antibody in any given assay. CST adapted the work by Uhlen, et. al., (“A Proposal for Validation of Antibodies.” Nature Methods (2016)) to build the Hallmarks of Antibody Validation, based on our decades of experience as an antibody manufacturer and our dedication to reproducible science.


CST Hallmarks of Antibody Validation

We guarantee that our antibodies are fit for purpose by carefully tailoring the combination of validation strategies applied to each product. 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.

Hallmark Strategy


Binary Model: Antibody signal is measured in model systems with known presence/absence of target signal. Includes wild-type vs. genetic knockout, targeted induction or silencing.
Ranged Expression: Antibody signal strength is measured in cell lines or tissues representing a known continuum of target expression levels. Includes siRNA and heterozygous knockout assays.
Orthogonal Data: Antibody signal is correlated to target expression in model systems measured using antibody independent assays. Includes mass spectrometry and in situ hybridization.
Multiple Antibodies: Antibody signal is compared to the signal observed using antibodies targeting nonoverlapping epitopes of the target. Includes IP, ChIP, and ChIP-seq.
Heterologous Expression: Antibody signal is evaluated in cell lines following heterologous expression of native (or mutated) target protein.
Complementary Assays: Antibody specificity may be validated using complementary assays. Includes competitive ELISA, peptide dot blots, peptide blocking, or protein arrays.