Ponceau S waste: Ponceau S staining for total protein normalization

Sander, Hannah; Wallace, Samantha; Plouse, Rachel; Tiwari, Shuchita; Gomes, Aldrin V.

doi:10.1016/j.ab.2019.03.010

Cited by 87 publications

(63 citation statements)

References 17 publications

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“…#2867, 1:1000 dilution) or the negative control anti-Hexokinase I (Cell Signaling #2024, 1:1000 dilution) at for 2 h, followed by anti-rabbit IgG, HRP-linked secondary antibody (Cell Signaling #7074, 1:1000 dilution) at for 2 h. Pierce ECL Plus western blotting substrate (Thermo) was used to detect targeted proteins using Typhoon 9400 Imager (Molecular Dynamics). The ImageJ software (National Institutes of Health, Bethesda, MD, USA) 63 was used to quantify the intensity of the bands of interest and normalized to Ponceau Stain 64 . Statistical tests on the densitometry values were performed using Welch’s t-test in R v.3.6.3.…”

Section: Methodsmentioning

confidence: 99%

Proteomic signatures of acute oxidative stress response to paraquat in the mouse heart

Dostal

Wood

Thomas

et al. 2020

Sci Rep

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The heart is sensitive to oxidative damage but a global view on how the cardiac proteome responds to oxidative stressors has yet to fully emerge. Using quantitative tandem mass spectrometry, we assessed the effects of acute exposure of the oxidative stress inducer paraquat on protein expression in mouse hearts. We observed widespread protein expression changes in the paraquat-exposed heart especially in organelle-containing subcellular fractions. During cardiac response to acute oxidative stress, proteome changes are consistent with a rapid reduction of mitochondrial metabolism, coupled with activation of multiple antioxidant proteins, reduction of protein synthesis and remediation of proteostasis. In addition to differential expression, we saw evidence of spatial reorganizations of the cardiac proteome including the translocation of hexokinase 2 to more soluble fractions. Treatment with the antioxidants Tempol and MitoTEMPO reversed many proteomic signatures of paraquat but this reversal was incomplete. We also identified a number of proteins with unknown function in the heart to be triggered by paraquat, suggesting they may have functions in oxidative stress response. Surprisingly, protein expression changes in the heart correlate poorly with those in the lung, consistent with differential sensitivity or stress response in these two organs. The results and data set here could provide insights into oxidative stress responses in the heart and avail the search for new therapeutic targets.

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Section: Methodsmentioning

confidence: 99%

Proteomic signatures of acute oxidative stress response to paraquat in the mouse heart

Dostal

Wood

Thomas

et al. 2020

Sci Rep

View full text Add to dashboard Cite

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“…Ponceau staining was performed for each blot to determine protein transfer and imaged. Ponceau S represented total protein and was used to normalize the gels for protein loading (26). The immunoblots were blocked in 5% nonfat dry milk in PBST (PBS containing 0.1% Tween 20) for 1 hour at room temperature and then followed by PBS washes; the blots were incubated with appropriate dilutions of primary antibodies overnight.…”

Section: Immunohistochemistry (Ihc)/ Immunofluorescence (If)mentioning

confidence: 99%

“…After subsequent washes in PBST, bound antibody was detected by chemiluminescence (Western Lightning Plus ECL, Perkin Elmer). Bands produced in the results were quantified using imageJ and normalized with ponceau S staining to confirm equal loading (26). Data was presented as relative intensity.…”

Section: Immunohistochemistry (Ihc)/ Immunofluorescence (If)mentioning

confidence: 99%

Quinomycin A reduces cyst progression in Polycystic Kidney Disease

Radadiya

Thornton

Magenheimer

et al. 2020

Preprint

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Polycystic kidney disease (PKD) is a genetic disorder that affects cilia homeostasis and causes progressive growth of tubular-derived cysts within the kidney. Efforts to find safer drugs for PKD have increased in the past few years after the successful launch of tolvaptan, the first approved drug to combat autosomal dominant PKD progression. Here we investigate the effects of Quinomycin A on progression of PKD. Quinomycin A is a bis-intercalator peptide that has previously shown to be effective against cancer progression. Quinomycin A treatment decreased cyst progression of human ADPKD primary renal epithelial cells grown in a 3D collagen gel to form cysts. In an orthologous mouse model of PKD, Quinomycin A administration reduced kidney to body weight ratios, and reduced cystogenesis. This was accompanied by decreased cell proliferation and fibrosis. Quinomycin treatments efficiently reduced the expression of Notch pathway proteins, RBPjk and HeyL in kidneys of PKD mice. Interestingly, Quinomycin treatments also normalized cilia lengths of collecting duct cyst-lining renal epithelia of PKD mice. This is the first preclinical study to our knowledge that demonstrates Quinomycin A has protective effects against PKD progression, in part by reducing Notch signaling and renal epithelial cilia lengths. Our findings suggest Quinomycin A has potential therapeutic value for PKD patients.

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“…Ponceau staining was performed for each blot to determine protein transfer and imaged. Ponceau S (total protein was used to normalize the gels for protein loading 57 ). The immunoblots were blocked in 5% nonfat dry milk in PBST (PBS containing 0.1% Tween 20) for 1 hour at room temperature and then followed by PBS washes; the blots were incubated with appropriate dilutions of primary antibodies overnight.…”

Section: Immunocytochemistry (Icc)mentioning

confidence: 99%

“…After subsequent washes in PBST, bound antibody was detected by chemiluminescence (Western Lightning Plus ECL, Perkin Elmer). Bands produced in the results were quantified using imageJ and normalized with ponceau S staining to confirm equal loading 57 . Data was presented as relative fold change.…”

Section: Immunocytochemistry (Icc)mentioning

confidence: 99%

Ciclopirox Olamine Induces Ferritinophagy and Ameliorates Disease Progression in Polycystic Kidney Disease

Radadiya¹,

Puri²,

Magenheimer³

et al. 2020

Preprint

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Despite the recent launch of Tolvaptan, the search for safer polycystic kidney disease (PKD) drugs continues. Ciclopirox, as the free acid (CPX) and its olamine salt (CPX-O) , are contained in number of commercially available topical antifungal agents. CPX is reported in the literature to possess anticancer activity in number of solid tumor cancers and hematological malignancy by several proposed mechanisms of action including chelation of iron and inhibition of iron dependent enzymes. Here, we show that CPX-O inhibited in vitro cystogenesis of primary human cyst epithelial cells cultured in 3D collagen matrix. To determine if CPX-O inhibits PKD progression, we treated PKD mice with a low dose of 10 mg/kg CPX-O by daily intraperitoneal injections from day 21 to day 49 post- partum. CPX-O reduced the kidney to body weight ratio of the PKD mice. This was associated with decreased cell proliferation decrease cystic area and improved renal function. We found that ferritin levels were significantly elevated in cystic kidneys of PKD mice, and that CPX-O treatment reduced renal ferritin levels and increased ferritinophagy marker, NCOA4. Our data suggest that CPX-O dose dependently induces ferritin degradation via ferritinophagy which is associated with decreased cyst growth and disease progression in PKD mice. Most importantly these data indicate that CPX-O, a drug used to treat skin infections and currently in clinical trials for cancer, has the potential to treat ADPKD.

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Ponceau S waste: Ponceau S staining for total protein normalization

Cited by 87 publications

References 17 publications

Proteomic signatures of acute oxidative stress response to paraquat in the mouse heart

Proteomic signatures of acute oxidative stress response to paraquat in the mouse heart

Quinomycin A reduces cyst progression in Polycystic Kidney Disease

Ciclopirox Olamine Induces Ferritinophagy and Ameliorates Disease Progression in Polycystic Kidney Disease

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