An Overview of Western Blotting for Determining Antibody Specificities for Immunohistochemistry

Abstract

Despite its overall simplicity, protein blotting or Western blotting has been proven to be a powerful procedure for the immunodetection of proteins, especially those that are of low abundance, following electrophoresis. The usefulness of this procedure stems from its ability to provide simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Protein blotting has evolved greatly since its inception and researchers have a variety of ways and means to carry out this transfer. This procedure is used in combination with other important antibody-based detection methods such as enzyme-linked immunosorbant assay and immunohistochemistry to provide confirmation of results both in research and diagnostic testing. Specificity of antibodies used for immunohistochemistry is of critical importance and therefore Western blot is a "must" to address antibodies' specificity.

Circulating Cell-Free DNA in Dogs with Mammary Tumors: Short and Long Fragments and Integrity Index

“…This is a critical step when testing antibodies produced against human antigens in a different species [31]. Considering their expression in our cohort of patients, Bcl-2 was more expressed in malignant tumors than in healthy tissue and in benign tumors, as already reported in CMTs [32,33] and in several human tumors [34–37].…”

Morgagnian cataract resulting from a naturally occurring nonsense mutation elucidates a role of CPAMD8 in mammalian lens development

“…However, the presumed absence of CPAMD8 in fetal and normal adult lenses was most likely only due to the reduced intensity of the immunohistochemical staining. Because, when using Western blotting as a more robust and quantitative method [31], a faint band at the expected size of CPAMD8 was also detectable in normal fetal and adult but not cataractous lenses (S1 Fig). So far there have been no reports of CPAMD8 in mammalian lens tissues, although it was detected in other ocular tissues and fluids e.g.…”

An overview of technical considerations for Western blotting applications to physiological research

“…The principle of the WB is the detection of protein(s) through the binding and recognition of antibodies (Ab) to one or more targets; this interaction should be highly specific between a portion of the antigen (protein) or epitope and the specific recognition sites found on the fragment antigen-binding (Fab) region of the antibody termed a paratope (Kurien et al 2011) (see Figure 1). The 1°Ab should be thoroughly assessed and validated to be specific and sensitive enough to detect the intended target protein.…”