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. 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.
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