Separation of membrane proteins by two‐dimensional electrophoresis using cationic rehydrated strips

Wenge, Birger; Bönisch, Heinz; Grabitzki, Julia; Lochnit, Günter; Schmitz, Brigitte; Ahrend, M. H. J.

doi:10.1002/elps.200700546

Cited by 15 publications

(7 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Exosomal proteins (700 μg/analysis/ duplicate) were subjected to two-dimensional electrophoresis or prehydrated plastic sheet gel strips for separation of hydrophobic proteins (29) followed by matrix-assisted laser desorption/ionization-time-of-flight analysis. Densitometric analyses were done with the PDQuest software after Flamingo staining (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

Cell Surface Tetraspanin Tspan8 Contributes to Molecular Pathways of Exosome-Induced Endothelial Cell Activation

et al. 2010

Self Cite

View full text Add to dashboard Cite

Tumor-derived exosomes containing the tetraspanin Tspan8 can efficiently induce angiogenesis in tumors and tumor-free tissues. However, little information exists on exosome-endothelial cell (EC) interactions or the proangiogenic role of tetraspanins, which are a constitutive component of exosomes. In this study, we used a rat adenocarcinoma model (AS-Tspan8) to explore the effects of exosomal Tspan8 on angiogenesis. Tspan8 contributed to a selective recruitment of proteins and mRNA into exosomes, including CD106 and CD49d, which were implicated in exosome-EC binding and EC internalization. We found that EC internalized Tspan8-CD49d complex-containing exosomes. Exosome uptake induced vascular endothelial growth factor (VEGF)-independent regulation of several angiogenesis-related genes, including von Willebrand factor, Tspan8, chemokines CXCL5 and MIF, chemokine receptor CCR1, and, together with VEGF, VEGF receptor 2. EC uptake of Tspan8-CD49d complex-containing exosomes was accompanied by enhanced EC proliferation, migration, sprouting, and maturation of EC progenitors. Unraveling these new pathways of exosome-initiated EC regulation could provide new options for therapeutic interference with tumor-induced angiogenesis.

show abstract

Section: Methodsmentioning

confidence: 99%

Cell Surface Tetraspanin Tspan8 Contributes to Molecular Pathways of Exosome-Induced Endothelial Cell Activation

et al. 2010

Self Cite

View full text Add to dashboard Cite

show abstract

“…Cytosolic or membrane-associated proteins (150 μg) were subjected to gel strips (pH 7–11 NL, 13 cm) in sample buffer [4% CHAPS, 30 mM DTT, 20 mM Tris-base, 2% IPG buffer, 1% bromophenol blue (added immediately before use)] and maintained 24 h at room temperature for rehydration. Then, strips were placed in a horizontal running tray for IEF and covered with mineral oil to prevent dehydration during electrophoresis ( Wenge et al, 2008 ). Electrophoresis was performed at 0–100 V (1 mA) for 5 h, 100–3500 V (1 mA) for 6 h and 3500 V (1 mA) for 6 h at 20°C.…”

Section: Methodsmentioning

confidence: 99%

Proteomic Analysis of 2,4,6-Trinitrotoluene Degrading Yeast Yarrowia lipolytica

2017

View full text Add to dashboard Cite

2,4,6-trinitrotoluene (TNT) is a common component of many explosives. The overproduction and extensive usage of TNT significantly contaminates the environment. TNT accumulates in soils and aquatic ecosystems and can primarily be destroyed by microorganisms. Current work is devoted to investigation of Yarrowia lipolytica proteins responsible for TNT transformation through the pathway leading to protonated Meisenheimer complexes and nitrite release. Here, we identified a unique set of upregulated membrane and cytosolic proteins of Y. lipolytica, which biosynthesis increased during TNT transformation through TNT-monohydride-Meisenheimer complexes in the first step of TNT degradation, through TNT-dihydride-Meisenheimer complexes in the second step, and the aromatic ring denitration and degradation in the last step. We established that the production of oxidoreductases, namely, NADH flavin oxidoreductases and NAD(P)+-dependent aldehyde dehydrogenases, as well as transferases was enhanced at all stages of the TNT transformation by Y. lipolytica. The up-regulation of several stress response proteins (superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase) was also detected. The involvement of intracellular nitric oxide dioxygenase in NO formation during nitrite oxidation was shown. Our results present at the first time the full proteome analysis of Y. lipolytica yeast, destructor of TNT.

show abstract

“…The 2D separation is achieved by the fact that membrane proteins migrate differentially in the presence of 16-BAC [18,19]. Wenge and coworkers developed a method based on rehydrated plastic sheet gel strips, which makes the 16-BAC approach easier to handle [20]. The 16-BAC technology has been applied to characterize the membrane proteome of yeast mitochondria [21], as well as the membrane proteome of immunopurified presynaptic compartments [22].…”

Section: Application Of Proteomics Approachesmentioning

confidence: 99%

Proteomics of total membranes and subcellular membranes

Groen

Lilley

2010

Expert Review of Proteomics

View full text Add to dashboard Cite

Membrane proteins are key molecules in the cell and are important targets for drug development. Much effort has, therefore, been directed towards research of this group of proteins, but their hydrophobic nature can make working with them challenging. Here we discuss methodologies used in the study of the membrane proteome, specifically discussing approaches that circumvent technical issues specific to the membrane. In addition, we review several techniques used for visualization, qualification, quantitation and localization of membrane proteins. The combination of the techniques we describe holds great promise to allow full characterization of the membrane proteome and to map the dynamic changes within it essential for cellular function.

show abstract

Separation of membrane proteins by two‐dimensional electrophoresis using cationic rehydrated strips

Cited by 15 publications

References 40 publications

Cell Surface Tetraspanin Tspan8 Contributes to Molecular Pathways of Exosome-Induced Endothelial Cell Activation

Cell Surface Tetraspanin Tspan8 Contributes to Molecular Pathways of Exosome-Induced Endothelial Cell Activation

Proteomic Analysis of 2,4,6-Trinitrotoluene Degrading Yeast Yarrowia lipolytica

Proteomics of total membranes and subcellular membranes

Contact Info

Product

Resources

About