Stain-Free total-protein normalization enhances the reproducibility of Western blot data

Maloy, A.; Alexander, Shaun; Andreas, Alexis; Nyunoya, Toru; Chandra, Divay

doi:10.1016/j.ab.2022.114840

Cited by 10 publications

(4 citation statements)

References 19 publications

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“…Total protein staining for the normalization of western blotting is recognized for its reliability and reproducibility, providing a consistent method to account for variability in protein loading and transfer across samples. All quantitative analyses were conducted using the original raw files produced by the LI-COR software, which analyses band intensity independently of the background, allowing for accurate comparisons between samples (Aldridge et al, 2008;Collins et al, 2015;Eaton et al, 2013;Ghosh et al, 2014;Maloy et al, 2022). with the main effects of diet (CON vs. PGR) and sex (male vs. female)…”

Section: Western Blottingmentioning

confidence: 99%

Postnatal growth restriction alters myocardial mitochondrial energetics in mice

Visker,

Leszczynski,

Wellette‐Hunsucker

et al. 2024

Experimental Physiology

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Postnatal growth restriction (PGR) can increase the risk of cardiovascular disease (CVD) potentially due to impairments in oxidative phosphorylation (OxPhos) within cardiomyocyte mitochondria. The purpose of this investigation was to determine if PGR impairs cardiac metabolism, specifically OxPhos. FVB (Friend Virus B‐type) mice were fed a normal‐protein (NP: 20% protein), or low‐protein (LP: 8% protein) isocaloric diet 2 weeks before mating. LP dams produce ∼20% less milk, and pups nursed by LP dams experience reduced growth into adulthood as compared to pups nursed by NP dams. At birth (PN1), pups born to dams fed the NP diet were transferred to LP dams (PGR group) or a different NP dam (control group: CON). At weaning (PN21), all mice were fed the NP diet. At PN22 and PN80, mitochondria were isolated for respirometry (oxygen consumption rate, ) and fluorimetry (reactive oxygen species emission, ) analysis measured as baseline respiration (LEAK) and with saturating ADP (OxPhos). Western blotting at PN22 and PN80 determined protein abundance of uncoupling protein 3, peroxiredoxin‐6, voltage‐dependent anion channel and adenine nucleotide translocator 1 to provide further insight into mitochondrial function. ANOVAs with the main effects of diet, sex and age with α‐level of 0.05 was set a priori. Overall, PGR (7.8 ± 1.1) had significant (P = 0.01) reductions in respiratory control in complex I when compared to CON (8.9 ± 1.0). In general, our results show that PGR led to higher electron leakage in the form of free radical production and reactive oxygen species emission. No significant diet effects were found in protein abundance. The observed reduced respiratory control and increased ROS emission in PGR mice may increase risk for CVD in mice.

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Section: Western Blottingmentioning

confidence: 99%

Postnatal growth restriction alters myocardial mitochondrial energetics in mice

Visker,

Leszczynski,

Wellette‐Hunsucker

et al. 2024

Experimental Physiology

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“…Immunocomplexes were detected using ECL detection kit NeoPRO Femto (NeoBiotech; Nanterre, France) and the quantification of the detected bands was related to the total proteins transferred to the membrane (stain-free blot). Quantification of both total protein (Stain-free blot) and detected bands was performed using the Chemidoc TM MP Imaging System (Biorad, Segrate, MI, Italy) and normalization was performed using Image Lab Software (v.6.0.0, Biorad, Segrate, MI, Italy) [91].…”

Section: Protein Extraction and Western Blot Assaymentioning

confidence: 99%

Herpes Simplex Virus ICP27 Protein Inhibits AIM 2-Dependent Inflammasome Influencing Pro-Inflammatory Cytokines Release in Human Pigment Epithelial Cells (hTert-RPE 1)

Caproni,

Nordi,

Fontana

et al. 2024

IJMS

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Although Herpes simplex virus type 1 (HSV-1) has been deeply studied, significant gaps remain in the fundamental understanding of HSV-host interactions: our work focused on studying the Infected Cell Protein 27 (ICP27) as an inhibitor of the Absent-in-melanoma-2 (AIM 2) inflammasome pathway, leading to reduced pro-inflammatory cytokines that influence the activation of a protective innate immune response to infection. To assess the inhibition of the inflammasome by the ICP27, hTert-immortalized Retinal Pigment Epithelial cells (hTert-RPE 1) infected with HSV-1 wild type were compared to HSV-1 lacking functional ICP27 (HSV-1∆ICP27) infected cells. The activation of the inflammasome by HSV-1∆ICP27 was demonstrated by quantifying the gene and protein expression of the inflammasome constituents using real-time PCR and Western blot. The detection of the cleavage of the pro-caspase-1 into the active form was performed by using a bioluminescent assay, while the quantification of interleukins 1β (IL-1β) and 18 (IL-18)released in the supernatant was quantified using an ELISA assay. The data showed that the presence of the ICP27 expressed by HSV-1 induces, in contrast to HSV-1∆ICP27 vector, a significant downregulation of AIM 2 inflammasome constituent proteins and, consequently, the release of pro-inflammatory interleukins into the extracellular environment reducing an effective response in counteracting infection.

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“…Routine steps of Ponceau S staining Though there are many articles and instructions for Ponceau S staining, not rare are cases where researchers complain about the poor performance of this method. We used "Ponceau S staining" as a keyword to search in PubMed and summarized the routine steps of this method (Table S7) [5,16,23,[43][44][45][46][47][48][49][50]. It is still unclear whether the membrane needs to be washed before dyeing, whether the experimental temperature needs to be strictly controlled, and what imaging method to use.…”

Section: 24mentioning

confidence: 99%

Western blot normalization: Time to choose a proper loading control seriously

Wang

Han

et al. 2023

Electrophoresis

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Recent research has questioned the validity of housekeeping proteins in Western blot. Our present study proposed new ideas for Western blot normalization that improved the reproducibility of scientific research. We used the Gene Expression Omnibus (GEO) database and the web tool GEO2R to exclude unstable housekeeping genes quickly. In ischemic heart tissues, actin and tubulin changed significantly, whereas no statistically significant changes were observed in the expression of genes relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Besides, the reliability of GAPDH was further examined by Western blot. Additionally, unstable housekeeping genes were found in other animal models of cardiovascular medicine. We also found that sodium dodecyl sulfate and temperature significantly impacted the results of Ponceau S staining. Membranes stained with Ponceau S after immunodetection could avoid this interference, and the coefficients of variation for post-immunodetection staining are lower than those produced by GAPDH immunodetection. Overall, we described a new use of differential gene expression analysis and proposed a modified Ponceau S staining method, which provided researchers with a proper loading control for Western blot and hence could improve reproducibility in research.

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Stain-Free total-protein normalization enhances the reproducibility of Western blot data

Cited by 10 publications

References 19 publications

Postnatal growth restriction alters myocardial mitochondrial energetics in mice

Postnatal growth restriction alters myocardial mitochondrial energetics in mice

Herpes Simplex Virus ICP27 Protein Inhibits AIM 2-Dependent Inflammasome Influencing Pro-Inflammatory Cytokines Release in Human Pigment Epithelial Cells (hTert-RPE 1)

Western blot normalization: Time to choose a proper loading control seriously

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