If one of the primary antibodies is derived from goat, whereas the other is derived from mice, the corresponding secondary antibodies must be anti-goat and anti-mouse secondary antibodies, respectively. However, the use of secondary antibodies from the same species as the primary antibodies should be avoided, especially in double-labeling experiments. Species source of the secondary antibody: there is usually no predictable connection between species source and the quality of the secondary antibody. If the type of the primary antibody is not clear, IgG against the corresponding species can be used. If the primary monoclonal antibody is of a certain subclass of mouse IgG (IgG1, IgG2a, IgG2b, or IgG3), then almost all anti-mouse IgG can bind to it, or the secondary antibody can be selected to specifically target this subclass. If the primary antibody is mouse IgM, then the corresponding secondary antibody should be anti-mouse IgM. Polyclonal antibodies are mainly IgG immunoglobulins, so the corresponding secondary antibodies are anti-IgG antibodies. This is usually applicable for monoclonal antibodies. Type of primary antibody: the secondary antibody must match the class or subclass of the primary antibody. For example, if the primary antibody is a mouse-derived monoclonal antibody, an anti-mouse secondary antibody (goat anti-mouse or rabbit anti-mouse) should be selected. 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. 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.

Western blot quantification imagej