Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G‐250 and R‐250

Neuhoff, Volker; Arold, Norbert; Taube, Dieter; Ehrhardt, Wolfgang

doi:10.1002/elps.1150090603

Cited by 2,486 publications

(1,467 citation statements)

References 6 publications

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“…Three biological replicates were analysed for each sample. For spot excision, gels were loaded with 100 µg (BSA equivalents) of proteins and stained with colloidal coomassie blue [28].…”

Section: Extracellular Protein Extractionmentioning

confidence: 99%

Investigating Aspergillus nidulans secretome during colonisation of cork cell walls

Martins

Garcia

Varela

et al. 2014

Journal of Proteomics

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Cork, the outer bark of Quercus suber, shows a unique compositional structure, a set of remarkable properties, including high recalcitrance. Cork colonisation by Ascomycota remains largely overlooked. Herein, Aspergillus nidulans secretome on cork was analysed (2DE).Proteomic data were further complemented by microscopic (SEM) and spectroscopic (ATR-FTIR) evaluation of the colonised substrate and by targeted analysis of lignin degradation compounds (UPLC-HRMS). Data showed that the fungus formed an intricate network of hyphae around the cork cell walls, which enabled polysaccharides and lignin superficial degradation, but probably not of suberin. The degradation of polysaccharides was suggested by the identification of few polysaccharide degrading enzymes (β-glucosidases and endo-1,5--L-arabinosidase).Lignin degradation, which likely evolved throughout a Fenton-like mechanism relying on the activity of alcohol oxidases, was supported by the identification of small aromatic compounds (e.g. cinnamic acid and veratrylaldehyde) and of several putative high molecular weight lignin degradation products. In addition, cork recalcitrance was corroborated by the identification of several protein species which are associated with autolysis. Finally, stringent comparative proteomics revealed that A. nidulans colonisation of cork and wood share a common set of enzymatic mechanisms. However the higher polysaccharide accessibility in cork might explain the increase of β-glucosidase in cork secretome.

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“…Three biological replicates were analysed for each sample. For spot excision, gels were loaded with 100 µg (BSA equivalents) of proteins and stained with colloidal coomassie blue [28].…”

Section: Extracellular Protein Extractionmentioning

confidence: 99%

Investigating Aspergillus nidulans secretome during colonisation of cork cell walls

Martins

Garcia

Varela

et al. 2014

Journal of Proteomics

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“…The second dimension was carried out on 9-16% polyacrylamide linear gradient gels (18 cm 9 20 cm 9 1.5 mm) at 10°C and 40 mA per gel constant current until the dye front reached the bottom of the gel. Analytical gels were stained with ammoniacal silver nitrate as previously described [33]; MS-preparative gels were stained with colloidal Coomassie [34].…”

Section: Sample Preparation and 2d Gel Electrophoresismentioning

confidence: 99%

Exploring the biochemical mechanisms of cytotoxic gold compounds: a proteomic study

et al. 2010

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We have recently shown that a group of structurally diverse gold compounds are highly cytotoxic toward a panel of 36 human tumor cell lines through a variety of biochemical mechanisms. A classic proteomic approach is exploited here to gain deeper insight into those mechanisms. This investigation is focused on Auoxo6, a novel binuclear gold(III) complex, and auranofin, a clinically established gold(I) antiarthritic drug. First, the 72-h cytotoxicity profiles of Auoxo6 and auranofin were determined against A2780 human ovarian carcinoma cells. Subsequently, protein extraction from gold-treated A2780 cells sensitive to cisplatin and 2D gel electrophoresis separation were carried out according to established procedures. Notably, both metallodrugs caused relatively modest changes in protein expression in comparison with controls as only 11 out of approximately 1,300 monitored spots showed appreciable quantitative changes. Very remarkably, six altered proteins were in common between the two treatments. Eight altered proteins were identified by mass spectrometry; among them was ezrin, a protein associated with the cytoskeleton and involved in apoptosis. Interestingly, two altered proteins, i.e., peroxiredoxins 1 and 6, are known to play crucial roles in the cell redox metabolism. Increased cleavage of heterogeneous ribonucleoprotein H was also evidenced, consistent with caspase 3 activation. Overall, the results of the present proteomic study point out that the mode of action of Auoxo6 is strictly related to that of auranofin, that the induced changes in protein expression are limited and selective, that both gold compounds trigger caspase 3 activation and apoptosis, and that a few affected proteins are primarily involved in cell redox homeostasis.

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“…After 2-DE, gels were scanned with a Typhoon TRIO scanner (GE Healthcare Bio-Sciences AB) using filters appropriate for each dye's excitation and emission wavelength. To obtain an adequate amount of the proteins from the individual spots for identification, 400 μg of the tumoral samples and 400 μg of the non-tumoral samples were separately run on 2D electrophoresis and were stained using a colloidal CBB G-250 procedure [11]. Preparative gels were scanned with an Image Scanner II (GE Healthcare Bio-Sciences AB).…”

Section: Two-dimensional Electrophoresis (2-de) and Image Analysismentioning

confidence: 99%

Proteomic Analysis of Differentially Expressed Proteins in Peripheral Cholangiocarcinoma

Darby

Vuillier-Devillers

Pinault

et al. 2010

Cancer Microenvironment

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Cholangiocarcinoma is an adenocarcinoma of the liver which has increased in incidence over the last thirty years to reach similar levels to other liver cancers. Diagnosis of this disease is usually late and prognosis is poor, therefore it is of great importance to identify novel candidate markers and potential early indicators of this disease as well as molecules that may be potential therapeutic targets. We have used a proteomic approach to identify differentially expressed proteins in peripheral cholangiocarcinoma cases and compared expression with paired non-tumoral liver tissue from the same patients. Two-dimensional fluorescence difference gel electrophoresis after labeling of the proteins with cyanines 3 and 5 was used to identify differentially expressed proteins. Overall, of the approximately 2,400 protein spots visualised in each gel, 172 protein spots showed significant differences in expression level between tumoral and non-tumoral tissue with p<0.01. Of these, 100 spots corresponding to 138 different proteins were identified by mass spectrometry: 70 proteins were over-expressed whereas 68 proteins were under-expressed in tumoral samples compared to nontumoral samples. Among the over-expressed proteins, immunohistochemistry studies confirmed an increased expression of 14-3-3 protein in tumoral cells while α-smooth muscle actin and periostin were shown to be overexpressed in the stromal myofibroblasts surrounding tumoral cells. α-Smooth muscle actin is a marker of myofibroblast differentiation and has been found to be a Ian A. Darby, Karine Vuillier-Devillers, and Émilie Pinault have equally contributed to this work.

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Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G‐250 and R‐250

Cited by 2,486 publications

References 6 publications

Investigating Aspergillus nidulans secretome during colonisation of cork cell walls

Investigating Aspergillus nidulans secretome during colonisation of cork cell walls

Exploring the biochemical mechanisms of cytotoxic gold compounds: a proteomic study

Proteomic Analysis of Differentially Expressed Proteins in Peripheral Cholangiocarcinoma

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