Common housekeeping proteins are upregulated in colorectal adenocarcinoma and hepatocellular carcinoma, making the total protein a better "housekeeper"

Hu, Xiaowen; Du, Shujiao; Yu, Jian; Yang, Xuhan; Yang, Chao; Zhou, Daizhan; Wang, Qingyu; Qin, Shengying; Yan, Xiaomei; Han, Dongmei; Wan, Chunling

doi:10.18632/oncotarget.11439

Cited by 25 publications

(30 citation statements)

References 31 publications

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“…In a systematic quantitative Western blot study, human colorectal adenocarcinoma (HCA) and hepatocellular carcinoma (HCC) were compared with adjacent noncancerous tissues. While the immunosignal intensities of four of five and two of seven tested HKPs differed significantly (in HCA and HCC tissue, respectively), Sypro Ruby‐stained total protein was equal and had very low CV% of ≤7.1%, in contrast to the HKPs (≥20%) …”

Section: Proposed Total Protein Stainings and Their Propertiesmentioning

confidence: 90%

“…Consequential issues : It is time‐consuming work. Spiking‐in equal amounts of a standard protein into each sample (similar to quantitative RNA analysis). Consequential issues : Spike‐in protein(s) must be chosen, acquired, and tested, which is time consuming. Visualizing the total protein of the gel before transfer onto the blotting membrane Visualizing the total protein of a parallel gel, either separated by molecular weight or stacked into a single band Visualizing the transferred total protein on the blotting membrane …”

Section: Alternatives To Traditional Loading Controlsmentioning

confidence: 99%

“…Visualizing the total protein of a parallel gel, either separated by molecular weight or stacked into a single band …”

Section: Alternatives To Traditional Loading Controlsmentioning

confidence: 99%

“…Likewise, α‐tubulin and β‐tubulin saturate above loading ranges of about 4 μg and 10 μg total protein of mouse ovary lysate and whole brain lysate (PVDF, chemiluminescence and fluorescence), respectively . Instead of signal saturation, other authors find a plateau in the range of ≈8 – 20 μ g total protein of colorectal adenocarcinoma lysate (PVDF, chemiluminescence). Other studies do not find saturation at such low quantities, but rather hyperbolic calibration curves under some conditions …”

Section: Introductionmentioning

confidence: 99%

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Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots

Moritz

2017

Proteomics

131

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Western blotting is an analytical method widely used for detecting and (semi-)quantifying specific proteins in given samples. Western blots are continuously applied and developed by the protein community. This review article focuses on a significant, but not yet well-established, improvement concerning the internal loading control as a prerequisite to accurately quantifying Western blots. Currently, housekeeping proteins (HKPs) like actin, tubulin, or GAPDH are often used to check for equal loading or to compensate potential loading differences. However, this loading control has multiple drawbacks. Staining of the total protein on the blotting membrane has emerged as a better loading control. Total protein staining (TPS) represents the actual loading amount more accurately than HKPs due to minor technical and biological variation. Further, the broad dynamic range of TPS solves the issue of HKPs that commonly fail to show loading differences above small loading amounts of 0.5-10 μg. Although these and further significant advantages have been demonstrated over the past 10 years, only a small percentage of laboratories take advantage of it. The objective of this review article is to collect and compare information about TPS options and to invite users to reconsider their applied loading control. Nine benefits of TPS are discussed and seven different variants are critically evaluated by comparing technical details. Consequently, this review article offers an orientation in selecting the appropriate staining type. I conclude that TPS should be the preferred loading control in future Western blot approaches.

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Section: Proposed Total Protein Stainings and Their Propertiesmentioning

confidence: 90%

Section: Alternatives To Traditional Loading Controlsmentioning

confidence: 99%

“…Visualizing the total protein of a parallel gel, either separated by molecular weight or stacked into a single band …”

Section: Alternatives To Traditional Loading Controlsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots

Moritz

2017

Proteomics

131

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“…Inappropriate endogenous controls can compromise the accuracy and reliability of the results. This is because these genes and proteins are commonly used to normalize data and correct experimental errors, thus allowing for direct comparisons of gene and/or protein expression [1].…”

Section: Introductionmentioning

confidence: 99%

Reference Gene and Protein Expression Levels in Two Different NAFLD Mouse Models

Araujo

Bordin

Carvalho

2020

Gastroenterology Research and Practice

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The expression levels of some reference genes and proteins are used for data normalization and quantification. However, these levels can change in response to experimental conditions or treatments. Aim. The aim of this work was to evaluate reference gene and protein expression in models of nonalcoholic fatty liver disease, using mice fed with a high-fat diet (HFD) and mice that are genetically obese (ob/ob). Main Methods. Histological staining techniques were used to verify the morphology and quantify the amount of lipid droplets present in the liver. Real-time polymerase chain reaction and immunoblotting were employed for monitoring protein expression and gene expression levels, respectively. Key Finding. The results showed that there was a substantial increase in the amount of lipid droplets in the livers of HFD and ob/ob animals when compared to the standard diet (SD) group. There was an observed reduction in the expression of β-actin (10%), α-tubulin (6%), GAPDH (19%), and RPL3 (15%) genes when comparing the ob/ob group to the HFD group. Additionally, the ob/ob mice displayed GAPDH protein levels that were substantially, but not significantly, reduced when compared to SD. Significance. It was concluded that there are slight differences in the expression levels of reference genes and proteins in these two NAFLD animal models, and researchers should consider these alterations when working with these models.

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Rapid detection of heat stress biomarkers in Atlantic salmon (Salmo salar) liver using targeted proteomics

Mendoza‐Porras,

Rusu,

Stratford

et al. 2023

Aquaculture Fish & Fisheries

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Most fish are ectothermic; therefore, their physiology is significantly affected by temperature. Aquaculture fish have limited ability to avoid elevated water temperatures, with impacts increasing as a result of climate change. To date, quantifying gene expression has been proposed to monitor heat stress in salmon liver. This study aimed to establish a faster multiplexed proteomics method to measure the abundance of thermal stress biomarkers in liver of salmon reared at 15°C or 20°C. Moreover, this study aimed to determine the effects that sample pooling, and data normalisation using housekeeping (HK) protein peptides would exert over the statistical significance of these thermal stress markers. A multiple reaction monitoring (MRM) mass spectrometry method, comprised 45 peptides derived from thermal stress markers and 10 peptides from HK proteins, was applied to measure these markers in liver of salmon reared at 15°C or 20°C. When samples were processed individually, 34 peptides were significant between salmon livers at 15°C or 20°C. In pooled samples, this decreased to five significant peptides. Peptides hprt1_HYADDLDR (hypoxanthine phosphoribosyl transferase) and gapdh_VPTPNVSVVDLTVR (glyceraldehyde‐3‐phosphate dehydrogenase) were the most stable and unstable HK protein peptides, respectively. When data was normalised with hprt1_HYADDLDR, 16 peptides were significant in individual samples and 13 in pooled samples. Significant peptides serpinh1a_ADLSNISGK, SerpinH1_TNSILFIGR, ela2_VVGGEDVR and gapdh_VPTPNVSVVDLTVR were common regardless of data strategy. A fast and reliable MRM method was established to validate thermal stress markers in salmon liver, where individual samples yielded better results than pooled samples. Sample pooling was only better when combined with normalisation as it validated twice the number of markers than sample pooling alone. This method could be applied to monitoring stress response in experiments involving feeding additives designed to mitigate thermal stress or in selective breeding programs to help understanding family variance in thermal tolerance.

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Common housekeeping proteins are upregulated in colorectal adenocarcinoma and hepatocellular carcinoma, making the total protein a better "housekeeper"

Cited by 25 publications

References 31 publications

Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots

Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots

Reference Gene and Protein Expression Levels in Two Different NAFLD Mouse Models

Rapid detection of heat stress biomarkers in Atlantic salmon (Salmo salar) liver using targeted proteomics

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