Western Blots versus Selected Reaction Monitoring Assays: Time to Turn the Tables?

Abstract

As of January 1, 2013, the paper entitled “Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications,” by Towbin and colleagues (1), had been cited 52,488 times (ISI Web of Knowledge v5.8), placing it among the elite group of papers that have truly transformed life science research. For more than 30 years, the nitrocellulose-based Western blotting technique introduced by this paper has been a principal method for the detection of specific proteins in complex biological samples. In the original paper, the authors already anticipated that refinements and variations of the basic technique could lead to the determination of properties of a protein other than its mere presence, and indeed such extensions have been exceedingly successful. The state of phosphorylation, the presence of other post-translational modifications, domain boundaries, estimation(s) of the molecular weight of the protein(s), and the approximate location(s) of antibody epitopes, among other important parameters, have been determined via Western blotting. However, these workers likely could not have anticipated that it would also become the de facto “gold standard” method for quantifying a protein in a complex sample. In fact, in the third sentence of their abstract (1) they wrote, “For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative,” suggesting that they considered accurate protein quantification by means of Western blotting to be a challenge.