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. 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.
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