Other Notable Methods of Membrane Protein Detection: A Brief Review

Kurien, Biji T.; Scofield, R. Hal

doi:10.1007/978-1-4939-2718-0_36

Cited by 5 publications

(5 citation statements)

References 35 publications

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“…The production of reactive oxygen species (ROS) in response to the activation of JNK-c-Jun pathways has been implicated in murine hepatocarcinogenesis 19 . Consistent with this finding, oxidized protein levels were significantly higher in C1485T-HBxTg mice than in WT-HBxTg mice, as assessed by OxyBlot 20 (Fig. 6a,b ).…”

Section: Resultssupporting

confidence: 84%

Contribution of C1485T mutation in the HBx gene to human and murine hepatocarcinogenesis

Hagiwara

Nishida

Park

et al. 2017

Sci Rep

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Although Hepatitis B virus (HBV) X gene mutations are frequently detected in HBV-related human hepatocellular carcinoma (HCC) patients, causative HBx mutations in the development of HCC have not yet been determined. We herein identified C1485T and C1653T mutations in the HBx gene as independent risk of HCC for HBV through the analysis using serum from chronic hepatitis B patients. We generated transgenic mice expressing wild-type (WT-HBxTg) and mutant (C1485T-HBxTg) HBx to assess the carcinogenic potential of mutated HBx. C1485T-HBxTg mice were more susceptible to diethylnitrosamine-induced hepatocarcinogenesis than WT-HBxTg mice and control non-Tg mice. The promotion of hepatocarcinogenesis in C1485T-HBxTg mice was accompanied by the activation of β-catenin and Jun N-terminal kinase (JNK) signaling pathways as well as the production of reactive oxygen species, whereas the activation of nuclear factor-kappa B in the livers of C1485T-HBxTg mice was attenuated. These results demonstrate that the HBx C1485T mutation contributes to human and murine hepatocarcinogenesis.

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Section: Resultssupporting

confidence: 84%

Contribution of C1485T mutation in the HBx gene to human and murine hepatocarcinogenesis

Hagiwara

Nishida

Park

et al. 2017

Sci Rep

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“…The most potential one regarding sensitivity and dynamic range may be Coomassie staining with a near-infrared detection [64] or staining with Ferrozine-ferrous complexes or Pyrogallol red-molybdate complexes [70]. Apart from the stainings listed in Table 2, several other membrane stainings have been published and may be tested as loading control [71]. Further TPS are continuously improved and invented, but some may fail as membrane stains.…”

Section: Other Potential Stainingsmentioning

confidence: 99%

Tubulin or not tubulin: Heading towards total protein staining as loading control in Western blots.

Moritz¹

2019

Preprint

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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 ten 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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“…Reasonable sensitivities and dynamic ranges may be obtained with Coomassie staining with a near‐infrared detection or staining with Ferrozine‐ferrous complexes or pyrogallol red‐molybdate complexes . Apart from the stainings listed in Table , several other membrane stainings have been published and may be tested as loading controls . Further TPSs are continuously discovered and developed, but some may fail as membrane stains.…”

Section: Other Potential Stainingsmentioning

confidence: 99%

“…[82] Apart from the stainings listed in Table 2, several other membrane stainings have been published and may be tested as loading controls. [83] Further TPSs are continuously discovered and developed, but some may fail as membrane stains. For example, a rapid staining method via dichlorofluorescein was recently reported to be more sensitive than silver staining and Sypro Ruby, [84] yet given that it reacts with the gel matrix (negative staining), it may not be applicable to stain membranes.…”

Section: Other Potential Stainingsmentioning

confidence: 99%

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.

show abstract

Other Notable Methods of Membrane Protein Detection: A Brief Review

Cited by 5 publications

References 35 publications

Contribution of C1485T mutation in the HBx gene to human and murine hepatocarcinogenesis

Contribution of C1485T mutation in the HBx gene to human and murine hepatocarcinogenesis

Tubulin or not tubulin: Heading towards total protein staining as loading control in Western blots.

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

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