Despite its overall simplicity, the Western Blot application is an effective procedure for the immunodetection of proteins and an essential tool within biological research. Nevertheless, proteins of the same length or similar size, such as isoforms of the same protein, are difficult to separate. With some adjustments, you can however achieve great results. Here we share some tips.
Western Blot (WB) is one of the most frequently antibody-based methods used in many fields of biochemical research. It is a simple way of detecting the presence or absence of a particular protein in a complex mixture, to compare protein levels, protein abundance, kinase activity, cellular localization, protein-protein interactions, or monitoring of post-translational modifications in different conditions or different tissues. If appropriately undertaken, WB is reliable, quantitative, both in relative and absolute terms, and extremely valuable (Bass et al., 2017).
WB combines the superior resolving power of polyacrylamide gel electrophoresis (PAGE) the specificity of antibodies, and the sensitivity of enzyme assays. The first step in WB is to prepare the protein sample by mixing it with the detergent called sodium dodecyl sulfate (SDS), which makes the proteins unfold into linear chains and coats them with a negative charge. Next, the protein molecules are separated according to their sizes using PAGE.
Following separation, the proteins are transferred from the gel onto a blotting membrane, usually, a nitrocellulose membrane, which binds and immobilizes the proteins followed by antigen detection by primary antibodies specific for the protein of interest, incubation with a secondary antibody linked to chemiluminescent or a fluorescent tag or label, develop, detection and quantification.