Misleading Westerns: Common Quantification Mistakes in Western Blot Densitometry and Proposed Corrective Measures

BioMed Research International
Trent A J ButlerJorge M Tolosa

Abstract

Densitometry data generated for Western blots are commonly used to compare protein abundance between samples. In the last decade, it has become apparent that assumptions underpinning these comparisons are often violated in studies reporting Western blot data in the literature. These violations can lead to erroneous interpretations of data and may contribute to poor reproducibility of research. We assessed the reliability of Western blot data obtained to study human myometrial tissue proteins. We ran dilution series of protein lysates to explore the linearity of densitometry data. Proteins analysed included αSMA, HSP27, ERK1/2, and GAPDH. While ideal densitometry data are directly proportional to protein abundance, our data confirm that densitometry data often deviate from this ideal, in which case they can fit nonproportional linear or hyperbolic mathematical models and can reach saturation. Nonlinear densitometry data were observed when Western blots were detected using infrared fluorescence or chemiluminescence, and under different SDS-PAGE conditions. We confirm that ghosting artefacts associated with overabundance of proteins of interest in Western blots can skew findings. We also confirm that when data to be normalised are...Continue Reading

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Citations

Jul 23, 2020·Journal of Mathematical Biology·Leonard SchmiesterJan Hasenauer
Apr 9, 2020·Frontiers in Cellular Neuroscience·Kathryn M MurphySimon Beshara
Nov 19, 2019·BioMed Research International·Shanshan LiuWeimin Chen
Nov 27, 2020·Biotechnology and Bioengineering·Christina SeisenbergerStefanie Wohlrab
Apr 11, 2021·Methods : a Companion to Methods in Enzymology·Loic LindnerGuillaume Pavlovic
Apr 24, 2021·Clinical Psychopharmacology and Neuroscience : the Official Scientific Journal of the Korean College of Neuropsychopharmacology·Cem CeritGürler Akpınar
Jun 5, 2021·Frontiers in Molecular Biosciences·Attila LehotzkyJudit Ovádi
Jul 30, 2021·Analytical Chemistry·Sahar IbrahimChristoph H Borchers
Jun 27, 2021·ChemMedChem·Shaymaa Emam Kassab, Samar Mowafy
Nov 19, 2021·Journal of Applied Physiology·Rachel C KelleyLeonardo F Ferreira

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Methods Mentioned

BETA
biopsies
Protein Assay
Electrophoresis
SMA

Software Mentioned

GE Imager
Studio Light
Image Quant TL
LI
MultiGauge
COR Image Studio
Image
Image Studio
Fujifilm Image Read LAS - 3000
GraphPad Prism

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