Species source of the primary antibody: the reactivity of the secondary antibody should be consistent with the species source of the primary antibody used. When selecting a secondary antibody, both the type of primary antibody and the requirements of subsequent detection schemes should be considered comprehensively: Usually, there are multiple secondary antibodies suitable for the subsequent detection of the target protein, and the selection can be optimized in specific experiments. If the results are highly correlated, then the antibody is validated for WB analysis. These methods include detection of whether the signal is eliminated or significantly reduced after genetic knockout or knockdown of the target gene analysis of the correlation between WB signals and signals of other detection methods (e.g., MS) in a set of different samples with variable expression of the target protein analysis of the correlation of protein levels by using two or more independent antibodies targeting different epitopes of the same protein expression of the target protein with a tag, and analysis of the correlation between antibody labeling and the detection of the tag. For unvalidated antibodies, there are suggested methodologies to validate the selectivity of the antibodies. There are currently databases that can be used for choosing characterized antibodies with high selectivities, such as Antibodypedia ( ), the Human Protein Atlas ( ), and the Antibody Registry ( ). The selectivity of antibodies directly affects WB results, and poor selectivity may lead to the misinterpretation of the results. This guide, therefore, aims to provide an updated and more concise and useable reference for future experiments and paper writing. Here, we will focus on some essential caveats during the WB experiment. However, concerns about WB have been voiced by many scientific journals in an effort to reduce potential mistakes and increase reproducibility. Therefore, WB remains the most commonly used methodology in the lab for protein detection. ELISA lacks loading controls, immunofluorescence is an in situ technique and is semiquantitative, while MS is expensive and depends on the experimental technique and conditions. Although there are many new alternative technologies, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and mass spectrometry (MS), they all have their own limitations to some extent. Any duplication or distribution of the information contained herein is strictly prohibited.Western blotting (WB) is an antibody-based experimental technique used to detect and quantify target proteins, which are often within a complex mixture extracted from cells or tissue. Links to other sites are provided for information only-they do not constitute endorsements of those other sites.
Western blot assay test professional#
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Western blot assay test series#
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