Blind spots on western blots: Assessment of common problems in western blot figures and methods reporting with recommendations to improve them

Abstract

Western blotting is a standard laboratory method used to detect proteins and assess their expression levels. Unfortunately, poor western blot image display practices and a lack of detailed methods reporting can limit a reader’s ability to evaluate or reproduce western blot results. While several groups have studied the prevalence of image manipulation or provided recommendations for improving western blotting, data on the prevalence of common publication practices are scarce. We systematically examined 551 articles published in the top 25% of journals in neurosciences (n = 151) and cell biology (n = 400) that contained western blot images, focusing on practices that may omit important information. Our data show that most published western blots are cropped and blot source data are not made available to readers in the supplement. Publishing blots with visible molecular weight markers is rare, and many blots additionally lack molecular weight labels. Western blot methods sections often lack information on the amount of protein loaded on the gel, blocking steps, and antibody labeling protocol. Important antibody identifiers like company or supplier, catalog number, or RRID were omitted frequently for primary antibodies and regularly for secondary antibodies. We present detailed descriptions and visual examples to help scientists, peer reviewers, and editors to publish more informative western blot figures and methods. Additional resources include a toolbox to help scientists produce more reproducible western blot data, teaching slides in English and Spanish, and an antibody reporting template.

Production of monoclonal antibodies against botulinum neurotoxin in Nicotiana benthamiana

“…Multiple bands on the blot can be caused by loading too much lysate onto a gel, which leads the detection antibody to bind nonspecifically to proteins in abundance. 68 A fully assembled IgG has a molecular weight of 150 kDa; we thereby conclude the presence of two heavy chains and two light chains linked together by disulfide bonds. 69 The plant-produced anti-BoNT mAbs were purified by protein A affinity chromatography, and their purity was determined by means of SDS-PAGE and a subsequent immunoblot.…”

Specific, sensitive and quantitative protein detection by in-gel fluorescence

“…Even seemingly insignificant differences in the procedure may have a surprisingly strong influence on the results 3 , 4 . Sadly, it is not uncommon in literature to just assume a linear relationship without sufficient controls or validations, resulting in misleading or incorrect interpretations 5 , 7 . In contrast, the in-gel fluorescence competition assay typically provides a linear signal ratio-to-substrate relationship, enabling accurate relative quantifications with little effort.…”

“…Overall, the procedure involves multiple steps, which are often adapted depending on the target protein, the chosen antibody combination, or the sample composition 1 , 2 . This impedes the comparability of different blots, in particular across different labs 3 – 5 . Even with identical samples and standard operating procedures, results from different users may vary significantly 6 .…”

Brain organoids engineered to give rise to glia and neural networks after 90 days in culture exhibit human-specific proteoforms

“…Currently, a majority of the BO literature uses immunofluorescence microscopy and RNA sequencing, which provide information on protein localization and levels of RNA transcripts, but does not provide information on protein processing (e.g., splice variants, post-translational modifications, cleavage) and does not accurately inform on protein levels unless volumetric analyses are conducted ( Wenzel et al, 2023a , c ). Thus, a wide comparison of cell and synapse markers between primary parenchymal tissues and BOs using immunoblotting would be beneficial, as it is the first step to determine whether BOs process proteins in a human-specific manner ( Kroon et al, 2022 ).…”