The Western blot (WB), also known as immunoblot, procedure has become a mainstay in supplemental testing since it readily permits visualization of the component antibody reactivities and, in essence, reveals the reason behind the reactivity in the screening assay ( 31, 32) ( Fig. Cowan, in AIDS and Other Manifestations of HIV Infection (Fourth Edition), 2004 Western Blot Dot-blot immunodetection, on the other hand, is an efficient alternative to traditional Western blots for high-throughput evaluation of protein expression using antibodies specific to common epitope tags ( Zeder-Lutz, Cherouati, Reinhart, Pattus, & Wagner, 2006). While immunoblotting provides insights into protein molecular weight and potential protein degradation issues, the technique is low throughput and not amenable to screening large enzyme libraries. Instead, readers are directed to useful resources describing immunoblotting for confirmation of protein expression in yeast systems ( Antebi & Fink, 1992 Fossati et al., 2015 Shen, Selvakumar, Stanford, & Hopper, 1993 Sherwood, Tsang, & Osley, 1993). Given the longstanding history and popularity of immunoblotting in protein biology, laboratory procedures are highly standardized ( Johnson, Gautsch, Sportsman, & Elder, 1984) and, therefore, are not detailed in this chapter. One of the most widely employed epitopes is the human influenza hemagglutinin (HA) tag comprised of the amino acid sequence YPYDVPDYA, required for recognition by the anti-HA antibody ( Wilson et al., 1984). The protein of interest is then detected through recognition by an antibody specific to the tag. A common approach entails the creation of a translational protein fusion consisting of the target protein expressed with a small epitope tag ( Kolodziej & Young, 1991). In the context of yeast BIA production, immunoblots have been utilized to confirm expression of a number of key BIA biosynthetic enzymes in yeast, including salutaridine synthase and a CPR ( Fig. Immunoblots, commonly referred to as Western blots, are widely employed to assess expression of heterologous proteins. Martin, in Methods in Enzymology, 2016 3.2.3 Immunoblotting
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