Western Blot: Illustrating the concepts of differential migration of proteins and band intensities as markers of protein abundance

Immunoblot is a commonly used biochemical technique for detecting the presence, and, in some cases, quantifying the amount of specific proteins; especially in mechanistic studies when a sequence or interacting molecular effectors participating in one or more pathways are of interest. Also known as Western blot, the assay separates proteins of different molecular weight via their differential migration under an applied electric field. Detection of protein presence could be effected by either general staining for protein (e.g., Ponceau S), or if the protein-of-interest is known, via antibodies. Antibody-mediated detection and quantification occurs in two steps: (i) primary antibody binding to the protein-of-interest; followed by (ii) use of either a fluorophore-conjugated or enzyme-coupled (e.g., horseradish peroxidase) secondary antibody specific to the primary antibody. Fluorescence intensity and enzyme activity could, in theory, be used for quantifying the relative abundance of different proteins. Nevertheless, problems with saturation, photobleaching etc. impacts on experiment reproducibility both within and across laboratories given the sensitivity of the technique to minutiae differences in a variety of experimental conditions such as buffer composition, incubation duration, and type of detection methods (specifically, electro-chemiluminescence and fluorescence). Indeed, the practice of Western blot is more art than science; though a recent systematic study (Jane, 2015) of various experimental variables that impact on assay accuracy and sensitivity would be useful in defining the operating envelope of this low throughput but targeted assay. The following slide illustrates the key concepts underlying immunoblots via defining, in a graphical sense, terms such as molecular weight ladder and loading control, as well as highlighting how band intensity relates to protein abundance and, most crucially, how migration along a polyacrylamide gel facilitates the separation of proteins of different molecular weights. Of note with respect to teaching is the abstraction of details such as inevitable differences in band shape and profiles; specifically, variation inherent in biological experiments and in loading of samples meant that bands on actual Western blots are seldom well-defined, straight or rectangular in profile. The slide should be useful for educational purposes or as a small panel in a larger slide.

Cited article: Kelvin A. Jane, 2015, “An analysis of critical factors for quantitative immunoblotting,” Science Signaling, Vol. 8, Issue, 371, doi: 10.1126/scisignal.2005966