Western blotting is a technique which involves separating a complex mixture of proteins (e.g., a cell lysate or tissue homogenate) on a gel before transferring them to a membrane for antibody-based detection. It can use either labeled primary antibodies for direct detection, or a combination of unlabeled primary antibodies and labeled secondary antibodies for indirect detection, and typically produces either a chemiluminescent or fluorescent readout. An advantage of direct detection is that it shortens the western blotting workflow by eliminating the need for a secondary antibody incubation step. However, indirect detection is more common due to the wide availability and relatively low cost of secondary antibodies for western blotting. An exception to this is when analyzing the products of immunoprecipitation (IP) reactions aimed at enriching a protein with a similar molecular weight to antibody heavy (50 kDa) or light (25 kDa) chains. In this scenario, direct detection is often preferred as it avoids the problem of secondary antibody reagents binding to the antibody used for target pulldown, which can obscure the band of interest. Horseradish peroxidase (HRP) conjugated secondary antibodies have historically been the most common secondaries used for western blotting, typically in combination with a chemiluminescent substrate based on luminol. Upon oxidation by HRP, the substrate forms an excited state product that subsequently emits light to return to its ground state. A similar approach is applied using Alkaline Phosphatase (AP) labeled secondary antibodies, which oxidize acridan- or 1,2-dioxetane-based substrates. In either scenario, the emitted light is detected either by exposing the blot to film in a dark room or with the use of a specialized reader. More recently, western blot detection has evolved to use antibodies labeled with fluorophores such as the Alexa Fluor®, Cyanine, DyLight®, FITC, and CF® dyes. A main advantage of fluorescent detection is that it allows for multiplexing without having to strip and re-probe the blot, which both takes time and risks damaging binding epitopes. This can be especially useful when there is a need to detect both total protein and a post-translationally modified isoform in the same experiment. In addition, fluorescent signals have a much longer lifetime (days to weeks) than chemiluminescence (minutes to hours) provided the blots are stored in the dark, allowing for further analysis at a later date should this be required.