Evaluating Strategies to Normalise Biological Replicates of Western Blot Data

Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

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PGE2 Modulates GABAA Receptors via an EP1 Receptor-Mediated Signaling Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000430143 ◽

2015 ◽

Vol 36 (5) ◽

pp. 1699-1711 ◽

Cited By ~ 2

Author(s):

Guang Yang ◽

Wen-Hao Dong ◽

Chang-Long Hu ◽

Yan-Ai Mei

Keyword(s):

Cell Surface ◽

Western Blot ◽

Gabaa Receptors ◽

Surface Expression ◽

Cell Surface Expression ◽

Granule Neurons ◽

Patch Clamp Technique ◽

Mammalian Tissues ◽

Ep1 Receptor ◽

Western Blot Data

Aims: PGE2 is one of the most abundant prostanoids in mammalian tissues, but its effect on neuronal receptors has not been well investigated. This study examines the effect of PGE2 on GABAA receptor currents in rat cerebellar granule neurons. Methods: GABAA currents were recorded using a patch-clamp technique. Cell surface and total protein of GABAA β1/2/3 subunits was carried out by Western blot analysis. Results: Upon incubation of neurons with PGE2 (1 µM) for 60 minutes, GABAA currents were significantly potentiated. This PGE2-driven effect could be blocked by PKC or CaMKII inhibitors as well as EP1 receptor antagonist, and mimicked by PMA or EP1 receptor agonist. Furthermore, Western blot data showed that PGE2 did not increase the total expression level of GABAA receptors, but significantly increased surface levels of GABAA β1/2/3 subunits after 1 h of treatment. Consistently, both PKC and CaMKII inhibitors were able to reduce PGE2-induced increases in cell surface expression of GABAA receptors. Conclusion: Activation of either the PKC or CaMKII pathways by EP1 receptors mediates the PGE2-induced increase in GABAA currents. This suggests that upregulation of postsynaptic GABAA receptors by PGE2 may have profound effects on cerebellar functioning under physiological and pathological conditions.

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ERRATUM: Author-Reported Correction of Western Blot Data: Kim JH, Park B, Gupta SC, Kannappan R, Sung B, and Aggarwal BB. Antioxid Redox Signal 16:413–427, 2012

Antioxidants and Redox Signaling ◽

10.1089/ars.2012.4968 ◽

2013 ◽

Vol 18 (2) ◽

pp. 219-220

Author(s):

Bharat B. Aggarwal

Keyword(s):

Western Blot ◽

Western Blot Data

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

BioMed Research International ◽

10.1155/2019/5214821 ◽

2019 ◽

Vol 2019 ◽

pp. 1-15 ◽

Cited By ~ 7

Author(s):

Trent A. J. Butler ◽

Jonathan W. Paul ◽

Eng-Cheng Chan ◽

Roger Smith ◽

Jorge M. Tolosa

Keyword(s):

Quality Control ◽

Western Blot ◽

Mathematical Models ◽

Total Protein ◽

Protein S ◽

Protein Abundance ◽

Western Blots ◽

Sds Page ◽

Control Protein ◽

Western Blot Data

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 not directly proportional to protein abundance, it is a mistake to use the normalisation technique of dividing densitometry data from the protein-of-interest with densitometry data from loading control protein(s), as this can cause the normalised data to be unusable for making comparisons. Using spiked proteins in a way that allowed us to control the total protein amount per lane, while only changing the amount of spiked proteins, we confirm that nonlinearity and saturation of densitometry data, and errors introduced from normalisation processes, can occur in routine assays that compare equal amounts of lysate. These findings apply to all Western blot studies, and we highlight quality control checks that should be performed to make Western blot data more quantitative.

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Nitidine chloride possesses anticancer property in lung cancer cells through activating Hippo signaling pathway

Cell Death Discovery ◽

10.1038/s41420-020-00326-7 ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Jing Zhang ◽

Linhui Wu ◽

Chaoqun Lian ◽

Shuo Lian ◽

Shimeng Bao ◽

...

Keyword(s):

Cell Cycle ◽

Western Blot ◽

Signaling Pathway ◽

Migration And Invasion ◽

Hippo Signaling ◽

Protein Expression Level ◽

Hippo Signaling Pathway ◽

Proliferation Ability ◽

And Migration ◽

Western Blot Data

Abstract Nitidine chloride (NC) has significant anti-tumor properties; however, the precise mechanism related to NC still needs further investigation. This study intends to investigate the anti-tumor functions and the feasible molecular basis of NC in NSCLC cells. Therefore, we determined the mechanism of NC-mediated anti-tumor function through various methods. Cell proliferation ability and migration and invasion were detected by CCK-8, colony formation assay and Transwell assay, respectively. Furthermore, flow cytometry was used to detect apoptosis, cell cycle and ROS. Moreover, protein expression level was measured by western blot. Our results showed that NC can inhibit the growth, motility of NSCLC cells, induce apoptosis and arrest cell cycle. Meanwhile, NC increased the level of ROS in NSCLC cells. Moreover, western blot data showed that NC suppressed the expression of Lats1, Mob1, and YAP, and enhanced the expression of p-Lats1, p-Mob1, p-YAP1 (ser127). Overall, our research reveals that NC exerts anticancer activity by activating and modulating the Hippo signaling pathway.

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Assembly Requirements and Role of CotH during Spore Coat Formation in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.181.8.2631-2633.1999 ◽

1999 ◽

Vol 181 (8) ◽

pp. 2631-2633 ◽

Cited By ~ 40

Author(s):

R. Zilhão ◽

G. Naclerio ◽

A. O. Henriques ◽

L. Baccigalupi ◽

C. P. Moran ◽

...

Keyword(s):

Bacillus Subtilis ◽

Western Blot ◽

Structural Component ◽

Ultrastructural Analysis ◽

Spore Coat ◽

Western Blot Data

ABSTRACT We report Western blot data showing that the 42.8-kDa product of the previously characterized cotH locus (8) is a structural component of the Bacillus subtilis spore coat. We show that the assembly of CotH requires both CotE and GerE. In agreement with these observations, the ultrastructural analysis of purified spores suggests that CotH is needed for proper formation of both inner and outer layers of the coat.

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Challenges of Interpreting Dystrophin Content by Western Blot

US Neurology ◽

10.17925/usn.2019.15.1.40 ◽

2019 ◽

Vol 15 (1) ◽

pp. 40 ◽

Cited By ~ 3

Author(s):

Frederick J Schnell ◽

Diane Frank ◽

Sue Fletcher ◽

Russell D Johnsen ◽

Steve D Wilton ◽

...

Keyword(s):

Clinical Trials ◽

Data Analysis ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Western Blot ◽

Becker Muscular Dystrophy ◽

Human Muscle ◽

Healthy Human ◽

Dystrophin Protein ◽

Western Blot Data

The Duchenne muscular dystrophy community has recently seen the first approved therapy for the restoration of dystrophin, based on its ability to increase levels of dystrophin protein, as determined by western blot. The approval, along with the initiation of clinical trials evaluating other dystrophin-restoring therapies, highlights the importance of accurate dystrophin quantitation. Nonoptimized western blot methods can reflect inaccurate results, especially in the quantitation of low dystrophin levels. A few key changes to standards and data analysis parameters can result in a low level of dystrophin (<0.5% of a healthy biopsy) being inaccurately interpreted as >20% of the levels reported in healthy human muscle. A review of the dystrophin western blot data on Duchenne and Becker muscular dystrophy biopsies is conducted, along with a thorough investigation of methodologies to quantify dystrophin.

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miR-21-5p protects IL-1β-induced human chondrocytes from degradation

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-019-1160-7 ◽

2019 ◽

Vol 14 (1) ◽

Cited By ~ 7

Author(s):

Hai Zhu ◽

Xin Yan ◽

Meng Zhang ◽

Feng Ji ◽

Shouguo Wang

Keyword(s):

Gene Expression ◽

Western Blot ◽

Protein Level ◽

Hyaline Cartilage ◽

Cartilage Regeneration ◽

Pathological Development ◽

Col2a1 Expression ◽

Oa Chondrocytes ◽

Disease Modifying ◽

Western Blot Data

Abstract Objective Osteoarthritis (OA) is a prevalent degenerative disease caused by various factors. MicroRNAs are important regulators in OA. MiR-21-5p expression is decreased in OA cartilage, but the effects of modulating miR-21-5p on cartilage regeneration are unknown. Therefore, our aim was to investigate the effects of miR-21-5p on cartilage metabolism of OA chondrocytes. Design We used IL-1β (10 ng/ml) to mimic OA chondrocytes. OA chondrocytes were transfected with miR-21-5p, the gene expression of COL2A1, MMP13, and ADAMTS5 was detected by qPCR. At the same time, COL2A1, MMP13, and ADAMTS5 were analyzed at the protein level by Western blot. CCK8 measured the cell’s viability and SA-β-gal detected the cell’s senescence. Results Upregulation of miR-21-5p had increased COL2A1 expression and decreased MM P13 and ADAMTS5 expression, which were in accord with Western blot data. SA-β-gal activity significantly increased, the viability was decreased in OA chondrocytes, and upregulation of miR-21-5p can decrease the SA-β-gal activity and increase cell viability. Conclusion MiR-21-5p might be a potential disease-modifying compound in OA, as it promotes hyaline cartilage production. These results provided that novel insights into the important function in OA pathological development.

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