How to Validate Antibodies

Friday, 31 January, 2020


  • validating antibodies
  • western blotting
  • antibody validation

    • “Describe data supporting antibody specificity, including post-translational modifications or neoepitopes”1

    Antibody vocabulary

    Before diving into antibody validation, there are a couple of terms you need to know first.

    • Antibody: an immunoglobulin protein that recognizes a specific antigen or target of interest
    • Antigen: any substance that can elicit an immune response
    • Epitope: the specific region of an antigen that the antibody recognizes
    • Paratope: the specific region of an antibody that recognizes and binds to the epitope
    • Specificity: recognition of the target antigen by the antibody
    • Selectivity: preferential binding of the antibody to the target antigen in a complex sample
    • Affinity: bond strength between the antibody’s paratope and the antigen’s epitope
    • Cross-reactivity or non-specific binding: binding of the antibody to other sample proteins with epitopes similar to the target protein

    Tips on validating a primary antibody

    Prior to starting your experiment, minimize any potential issues by validating your antibodies. Using the wrong antibody could create false-positive or false-negative results on which further experimentation is based. Consequently, it is important to always validate your antibody upon receipt, as common sources of variability include shipping complications, fluctuations in temperature, and even storage conditions.

    Experimental aspects such as incubation temperature, primary antibody dilution, and choice of membrane and blocking buffer can also influence antibody binding. To help mitigate issues, follow the tips below to validate your primary antibodies:

    • Use positive and negative controls to confirm that the band intensity in your membrane changes accordingly. Use knockout strategies to decrease target abundance or growth factors to induce or inhibit target expression.
    • Record all information. This includes the antibody name and source, as well as the lot, catalog and clone number. Also consider keeping track of the assay conditions during antibody validation. Having these details organized and ready will make your publishing process much easier.
    • Demonstrate antibody specificity and selectivity. Ensure you have sufficient evidence to indicate that the antibody has recognized your target. For example, the antibody should recognize the target in a positive control, and the target band should be detected at the expected molecular weight. Selectivity can be easily influenced by the concentration of the target protein, so endogenous levels of target expression should also be demonstrated.

    Tips for two-color fluorescent Westerns

    • Choose primary antibodies raised in two different host species, if your target proteins are similar in molecular weight, so secondary antibodies can easily distinguish them
    • Choose secondary antibodies raised from the same host species to avoid cross-reactivity
    • Use highly cross-adsorbed antibodies such as IRDye® Secondary Antibodies

    For more information on how to validate your antibodies, check out the “Antibody Validation” learning course in the Lambda U® education portal.


    Reference:

    1. Collecting and Presenting Data.The Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology. Web. 24 September 2018.

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