Western blotting is probably the most applied method in contemporary cell biology. Invented in 1979 (see the original paper), western blotting is used to obtain the relative quantification of an individual protein from a complex mixture of other proteins. There are two main steps: First, separate the complex protein mixture by size (typically by SDS-PAGE), and second, detect the protein of interest using an antibody. It's a simple idea and it normally works.
It's also an extremely analogue technique. It's low throughput, delicate and manual. It requires tweezers, precision, and up until recently - photographic film (seriously). My girlfriend - chronically aware of my culinary incompetence - was shocked to discover my proficiency at folding filo pastry. 10 years of western blotting had yielded a transferable skill: Vegetable Samosas.
So it's fiddly and slow. But the real problem with western blotting is its dependence on biology. A blot is only ever as good as its primary antibody. Commercial antibody reagents are stochastically derived from an in vivo humoral immune response and each antibody is different. Some antibodies are great, many antibodies are crap. If you're working with a slightly obscure protein, the chances are your experiments will be limited by the availability of a good antibody.
In his book “The Nature of Technology”, W. Brian Arthur notes that as technologies age, they become "stretched". That is, modest additions "stretch" the performance of technology towards gradual improvement. This is distinct from true innovation - whereby tools from an alternative "domain" facilitate a dramatic change in a technology (see this old post for more). The western blot is extremely stretched. We've had fluorescent detection, improved transfer procedures and micro-westerns. All modest additions that make western blotting a little better and provide the illusion that the technique is modern.
Western blots have served the scientific community well. Our current knowledge of cell biology is indebted to their competence. But despite modest technical improvements over the years, western blotting will always be dependent on stochastically derived antibodies of variable quality. It's the same problem Towbin et al faced in 1979.
So after 34 years isn't it time we transitioned from the western blot?
Writing in MCP, Ruedi Aebersold certainly think so. As a pioneer of targeted mass-spectrometry Aebersold ponders: "Western Blots vs. SRM Assays: Time to turn the tables?" (edited for brevity):
"Recently, targeted proteomic methods, specifically selected reaction monitoring (SRM) have become prevalent. The method is conceptually similar to Western blotting. Both use assays that must be developed for each target protein to detect and quantify specific, predetermined (sets of) analytes in complex samples. However, the methods differ substantially in their implementation, the reliability of the resulting assays and the quality of results they produce. A Western blotting assay essentially depends on the specificity of the antibody used. In contrast, a SRM assay depends on multiple parameters, such as the retention time, the mass-to-charge ratio of the precursor ion and selected fragment ions of the targeted peptide and the relative signal intensities of the detected fragment (transition) signals. These values are then weighted and combined to derive a score that indicates the probability that the targeted peptide has been detected."
So SRM is a great technique. A method of the year even. But Aebersold's contention is not the application of SRM, but the perceived superiority of western blotting over SRM:
"Authors who submit papers containing quantitative protein data generated by MS are frequently asked by reviewers to validate some of the values by Western blotting. We believe with the advances that have occurred that this request is now outdated."
Independent of their respective merits, more people are familiar with western blots than with mass-spec data. Thus, when journal reviewers see mass-spec data, they sometimes ask for western blot 'validation' of the induced conclusions. They want researchers to use a worse technique to validate a better one. Think about that for a second. It's like asking a mathematician to check their results on an abacus incase their calculator is wrong.
Sentimentally isn't an easy bedfellow of rationality. Alas, the backlash:
"We posit that the request to validate quantitative MS data by Western blotting is no longer justified. In fact, considering that the vast majority of protein identifications claimed from biological samples are still derived from Western blotting, it may be time to ‘turn the tables’ and to request that Western blotting results, or at least the assays that support these results, be validated by MS."
Better start clearing out the darkroom. The triple-quads are coming.