![]() But first, it’s useful to understand how we got to this point. In this article we introduce some of these groundbreaking initiatives taken by Bio-Rad Laboratories. Fortunately, initiatives are under way to both improve antibody quality and arm researchers with viable tools to routinely assess antibody performance. When researchers investigate why their western blots failed, nearly 2 in 3 found that the primary antibody was at fault, according to a Bio-Rad surveyĭespite these issues, antibodies remain a critical tool for biology labs avoiding them is not an option.Researchers have said that 50% of their antibodies were of insufficient quality, even though they cite quality as the most important purchasing criteria, according to a research report by 1DegreeBio.Scientists typically have to test between 2 and 4 different products before they find an antibody that works well for their application.Manufacturing standards and quality control vary wildly between vendors, targets, and even between antibody lots. How many more stories like this one are out there waiting to be uncovered? Despite decades of commercial production and distribution, antibodies are still perceived as unreliable. The problem is: These antibodies remain on the market today and are sold with the same performance claims. They also challenged the dogma that Epo-R was widely expressed in many tissue types, including renal cells ( Elliott S, et al. 2010).Įlliott’s team went on to invalidate several of the most popular Epo-R antibodies in peer-reviewed publications. Unsurprisingly, no effects were observed in these clinical trials ( Endre Z, et al. As a result, their use of nonspecific antibodies spawned a flood of flawed results, which in turn led to the launch of new clinical trials ( Endre Z 2014). Still, they failed to use controls, especially negative controls to detect false-positive data, leaving themselves open to confirmation bias (that is, seeing what they wanted to see). Steven Elliott, PhD, the first author of the article published in Blood, does not believe the researchers using anti-Epo-R antibodies were ill-intentioned. One red flag was the molecular weight of the proteins, which ranged from 66 to 78 kDa Epo-R has an estimated size of 54 to 55 kDa.įormer Scientific Executive Director at Amgen 2007) - that, in many cases, the Epo-R bands “detected” were in fact a heat shock protein (HSP70). A convincing argument was presented - and has since been supported by other groups ( Brown WM, et al. Some of the cross-reactivity involved proteins specifically associated with tumor cells. They demonstrated that the Epo-R antibodies exhibited significant off-target binding. Unfortunately, the authors of the Blood article were right. ![]() If the antibodies were flawed, those published findings might also be mistaken. This body of research suggested that Epo-R was widely expressed in the cells of a range of tissue types, including tumor cells. Casting doubt on these antibodies was alarming, as an entire body of research rested on their validity. 2006) called into question the specificity of antibodies claiming to bind human erythropoietin receptors (Epo-R). For example, a paper in the journal Blood ( Elliott S, et al. However there are significant challenges associated with these crucial reagents, and many published research findings have been reevaluated because of antibodies. It is hard to imagine protein research without antibodies to facilitate immunodetection. 440:186-8.Antibodies are extremely important tools for life science research. (2013) Stain-Free total protein staining is a superior loading control to β-actin for Western blots. Anal Biochem. (2014) The necessity of and strategies for improving confidence in the accuracy of western blots. Gilda JE, Gomes AV. (2015) Western blotting using in-gel protein labeling as a normalization control: stain-free technology. Methods Mol Biol. Gilda JE, Ghosh R, Cheah JX, West TM, Bodine SC, Gomes AV. (2015) Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS). PLoS One. Mishra M, Tiwari S, Gomes AV. (2017) Protein purification and analysis: next generation Western blotting techniques. Expert review of proteomics. (2019) Ponceau S waste: Ponceau S staining for total protein normalization. Sander H, Wallace S, Plouse R, Tiwari S, Gomes AV. (2020) Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly-horseradish peroxidase (PolyHRP). Mishra M, Tiwari S, Gunaseelan A, Li D, Hammock BD, Gomes AV. Useful Western Blotting References from the Gomes Lab
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