Assay-specific validation should confirm that the primary antibody is specific for its target antigen and that it selectively binds its target in the presence of other antigens. To ensure reproducible results, it is important to evaluate antibodies within the intended assays and experimental contexts. Even within one type of assay, small differences in assay conditions (intentional or unintentional) can affect antibody performance ( 8, 14, – 17). Recommendations for antibody validation are different for each type of immunoassay, and an antibody that performs well in one assay, such as a Western blotting, might not be suitable for another assay ( 10, – 13). Unfortunately, antibody performance can vary considerably between suppliers and even batches.Īntibody specificity and selectivity are highly dependent on the particular assay context and can be difficult to predict.
![protein ladder on western blot protein ladder on western blot](https://www.jacksonimmuno.com/secondary-antibody-resource/wp-content/uploads/blotting-tank.jpg)
Well-characterized antibodies that consistently perform as expected are therefore essential for robust, reproducible research. The Western blotting process relies on two key properties of primary antibodies: specificity, an antibody's ability to recognize and bind to its target antigen and selectivity, an antibody's preference to bind its target antigen in the presence of a heterogeneous mixture of competing sample proteins. In addition to improving the standardization of antibodies, antibody performance is another common source of variability in Western blotting. MIAPAR-compliant data include information such as the production/purification process, experimental evidence, updated protocols, and other relevant details ( 7, 9). The international community has also come up with a proposal called Minimum Information About a Protein Affinity Reagent (MIAPAR), which aims to establish a stronger connection between antibody producers and users. More recently, the National Institutes of Health (NIH) released new guidelines for grant submission that requires investigators to describe how they will “ensure the identity and validity of key biological resources,” including antibodies (NOT-OD-18-228). In 2016, the International Working Group for Antibody Validation (IWGAV) 3 proposed guidelines for improving standards for antibody use and validation ( 5). Several peer-reviewed journal editors have commented on issues related to the lack of reproducible antibodies in life science research ( 8), and multiple initiatives are trying to look at ways in which antibodies can be standardized, including evaluation, protocols, and documentation. In the life sciences, use of accepted standards can promote reproducibility by developing consensus-based methods that reduce unintentional differences between experiments, as well as improving data reporting practices to increase awareness of intentional differences between experiments ( 4, – 7). The lack of a unifying framework or set of standards is a clear barrier to reproducibility. Many of the issues above arise from a common cause: researchers perform similar assays in many different ways. Sources of irreproducible research include incomplete reporting of experimental details, lack of reagent validation and controls, differences in analytic techniques, or measurements and differences in the interpretation of results ( 3, 4). Despite this, however, the reproducibility of research findings has been a growing concern ( 1, 2). They help to confirm that experimental observations are meaningful and reflect a biological truth when combined with robust statistical analysis. Repeated observations are essential to the scientific method. Routine implementation of standardized antibody validation and reporting in immunoassays such as Western blotting may promote improved reproducibility across the global life sciences community. The antibody should produce reproducible results within and between Western blotting experiments and the observed effect confirmed with a complementary or orthogonal method. Practical strategies are proposed for the validation of primary antibody specificity, selectivity, and reproducibility using Western blot analysis. Because the performance of primary antibodies is strongly influenced by assay context, recommendations for validation and usage are unique to each type of immunoassay.
![protein ladder on western blot protein ladder on western blot](https://www.bio-rad.com/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/protein_electrophoresis/technology_detail/pet141_recmbnt_ppp.png)
![protein ladder on western blot protein ladder on western blot](https://media.biocompare.com/m/37/article/588639/588639_hero.jpg)
The current lack of clear, accepted standards for antibody validation and reporting of experimental details contributes to this problem. Well-characterized antibody reagents play a key role in the reproducibility of research findings, and inconsistent antibody performance leads to variability in Western blotting and other immunoassays.