Pitfalls of Western Blots

Western blots are one of a category of images that involve densitometry, or measurement of brightness and darkness ratios, a category that dominates the field in image-related article retractions and corrections.   In Scientific Imaging with Photoshop, author Jerry Sedgewick states that, “In general, images destined for OD/I measurements should not be altered in any way.  Exceptions to that rule are acceptable as long as the procedures are documented and described in the publication.”   Images captured for the purpose of densitometry permit the least manipulation, as Jerry observes,  “The only permissible change to electrophoretic specimens, aside from those taken to conform image to outputs, is to eliminate dust and scratches, but only by a global application of a filter.”

Why then,  are Western blots the focus of so many retractions?  In a 2009  article, “Quantifying Western blots: Pitfalls of densitometry”,  Gassman and colleagues observe, “Although Western blots are frequently quantified, densitometry is not documented.”   In their study of 100 randomly selected papers, “none provided sufficient information on how Western blot results were translated into statistical values”.  The study concludes that “the necessity of clear definition and documentation of densitometry is evident from our analysis”  and adds that, “the same data can be used to make – statistically proven – completely contradictory statements.”

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