Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies
TL;DR: Under standard assay conditions, the ratio of the absorbances, 590 nm over 450 nm, is strictly linear with protein concentration, and a linear equation that perfectly fits the experimental data is provided on the basis of mass action and Beer's law.
About: This article is published in Analytical Biochemistry.The article was published on 1996-05-01. It has received 1003 citations till now. The article focuses on the topics: Bradford protein assay & Bicinchoninic acid assay.
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TL;DR: The increase in biomass yields and changes in other constituents indicated the influence ofsalinity and the organism's adaptability to the tested levels of salinity (17 mM to 85 mM).
414 citations
Cites methods from "Linearization of the Bradford Prote..."
...Protein content in the cell free (spent) medium was analysed by Bradford protein assay (Zor and Selinger, 1996)....
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TL;DR: By the analysis of fluorescence spectrum and fluorescence intensity, it was showed that BCPT has a strong ability to quench the intrinsic fluorescence of both bovine serum albumin and human serumalbumin through a static quenching procedure.
337 citations
TL;DR: These results indicate that unlike Smac/DIABLO, Omi/HtrA2's catalytic cleavage of IAPs is a key mechanism for it to irreversibly inactivate I APs and promote apoptosis.
Abstract: Omi/HtrA2 is a mitochondrial serine protease that is released into the cytosol during apoptosis to antagonize inhibitors of apoptosis (IAPs) and contribute to caspase-independent cell death. Here, we demonstrate that Omi/HtrA2 directly cleaves various IAPs in vitro, and the cleavage efficiency is determined by its IAP-binding motif, AVPS. Cleavage of IAPs such as c-IAP1 substantially reduces its ability to inhibit and ubiquitylate caspases. In contrast to the stoichiometric anti-IAP activity by Smac/DIABLO, Omi/HtrA2 cleavage of c-IAP1 is catalytic and irreversible, thereby more efficiently inactivating IAPs and promoting caspase activity. Elimination of endogenous Omi by RNA interference abolishes c-IAP1 cleavage and desensitizes cells to apoptosis induced by TRAIL. In addition, overexpression of cleavage-site mutant c-IAP1 makes cells more resistant to TRAIL-induced caspase activation. This IAP cleavage by Omi is independent of caspase. Taken together, these results indicate that unlike Smac/DIABLO, Omi/HtrA2's catalytic cleavage of IAPs is a key mechanism for it to irreversibly inactivate IAPs and promote apoptosis.
324 citations
Cites methods from "Linearization of the Bradford Prote..."
...The protein concentrations were determined by the modified Bradford method (Zor and Selinger 1996)....
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TL;DR: ROS-dependent potentiation of stress kinase pathways accounts for the sensitization of transformed cells to stress and anticancer drugs, and is shown to be independent of the activity of the particular transforming oncoprotein.
Abstract: Many primary tumors as well as transformed cell lines display high sensitivity to chemotherapeutic drugs and radiation. The molecular mechanisms that underlie this sensitivity are largely unknown. Here we show that the sensitization of transformed cells to stress stimuli is due to the potentiation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways. Activation of these pathways by the antitumor drug cis-platin (CDDP) and by other stress agents is markedly enhanced and is induced by lower stress doses in NIH 3T3 cells overexpressing epidermal growth factor receptor, HER1–2 kinase, or oncogenic Ras than in nontransformed NIH 3T3 cells. Inhibition of stress kinase activity by specific inhibitors reduces CDDP-mediated cell death in transformed cells, whereas overactivation of stress kinase pathways augments cells death. Potentiation of stress kinases is a common feature of cells transformed by different oncogenes, including cells derived from human tumors, and is shown here to be independent of the activity of the particular transforming oncoprotein. We further show that the mechanism that underlies potentiation of stress kinases in transformed cells involves reactive oxygen species (ROS), whose production is elevated in these cells. JNK/p38 activation is inhibited by antioxidants and in particular by inhibitors of the mitochondrial respiratory chain and NADPH oxidase. Conversely, by artificially elevating ROS levels in nontransformed NIH 3T3 cells we were able to induce potentiation of JNK/p38 activation. Taken together, our findings suggest that ROS-dependent potentiation of stress kinase pathways accounts for the sensitization of transformed cells to stress and anticancer drugs.
315 citations
TL;DR: The authors showed that Ustilago maydis is recognized early and triggers defence responses, whereas many of these early response genes are downregulated at later time points, whereas several genes associated with suppression of cell death are induced.
Abstract: The fungal pathogen Ustilago maydis establishes a biotrophic relationship with its host plant maize (Zea mays). Hallmarks of the disease are large plant tumours in which fungal proliferation occurs. Previous studies suggested that classical defence pathways are not activated. Confocal microscopy, global expression profiling and metabolic profiling now shows that U. maydis is recognized early and triggers defence responses. Many of these early response genes are downregulated at later time points, whereas several genes associated with suppression of cell death are induced. The interplay between fungus and host involves changes in hormone signalling, induction of antioxidant and secondary metabolism, as well as the prevention of source leaf establishment. Our data provide novel insights into the complexity of a biotrophic interaction.
274 citations
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TL;DR: The conclusion is drawn that the assay conditions have been well optimized by earlier workers, and the potential for improvement of the CBB protein assay is discussed.
66 citations