The Experts below are selected from a list of 23448 Experts worldwide ranked by ideXlab platform
George Iliakis - One of the best experts on this subject based on the ideXlab platform.
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application of alkaline Sucrose Gradient centrifugation in the analysis of dna replication after dna damage
Methods of Molecular Biology, 2009Co-Authors: Sascha Raschke, Jun Guan, George IliakisAbstract:Sucrose density Gradient ultracentrifugation is a powerful technique for fractionating macromolecules like DNA, RNA, and proteins. For this purpose, a sample containing a mixture of different size macromolecules is layered on the surface of a Gradient whose density increases linearly from top to bottom. During centrifugation, different size macromolecules sediment through the Gradient at different rates. The rate of sedimentation depends, in addition to centrifugal force, on the size, shape, and density of the macromolecules, as well as on the density and viscosity of the Gradient. In this way, macromolecules are separated by size with larger ones sedimenting towards the bottom and lighter ones remaining close to the top of the Gradient. The method has been particularly successful in the size fractionation of large DNA molecules and has been extensively used to measure induction and repair of DNA breaks after exposure to clastogenic factors. Here, we describe an adaptation of this method that can be used in the analysis of newly synthesized DNA formed during DNA replication. Through size analysis of nascent DNA in alkaline Sucrose Gradients, variations in replication activity can be measured after exposure of cells to DNA-damaging agents. The method is particularly useful as it allows distinction between DNA damage-mediated effects on chain elongation vs. replicon initiation, which is essential for an in-depth analysis of the intra-S-phase checkpoint. This ability makes the technique unique and justifies its somewhat labour-intensive nature.
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comparison of dna double strand break rejoining as measured by pulsed field gel electrophoresis neutral Sucrose Gradient centrifugation and non unwinding filter elution in irradiated plateau phase cho cells
International Journal of Radiation Biology, 1991Co-Authors: George Iliakis, D Blocher, Lorenz Metzger, Gabriel E PanteliasAbstract:SummaryThe initial (up to 30 min) rate of DNA double-strand break (dsb) rejoining was measured in irradiated plateau-phase CHO cells, in a set of parallel experiments using the same cell suspension, by means of non-unwinding filter elution, neutral Sucrose Gradient centrifugation, and two pulsed-field gel electrophoresis assays: asymmetric field inversion gel electrophoresis (AFIGE) and clamped homogeneous electric field (CHEF) gel electrophoresis. The rate of DNA dsb rejoining was compared to the rate of rejoining of chromatin breaks measured, also in the same cell population, using the technique of premature chromosome condensation (PCC). Two radiation exposures, 25 Gy and/or 50 Gy, were used and applied to the individual parts of the experiments according to the sensitivity of the assay under investigation. Similar values for the initial rate of DNA dsb rejoining were obtained with all assays used, with t1/2 ranging between 10 and 12 min after exposure to 25 Gy and between 15 and 20 min after exposure ...
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comparison of dna double strand break rejoining as measured by pulsed field gel electrophoresis neutral Sucrose Gradient centrifugation and non unwinding filter elution in irradiated plateau phase cho cells
International Journal of Radiation Biology, 1991Co-Authors: George Iliakis, D Blocher, Lorenz Metzger, Gabriel E PanteliasAbstract:The initial (up to 30 min) rate of DNA double-strand break (dsb) rejoining was measured in irradiated plateau-phase CHO cells, in a set of parallel experiments using the same cell suspension, by means of non-unwinding filter elution, neutral Sucrose Gradient centrifugation, and two pulsed-field gel electrophoresis assays: asymmetric field inversion gel electrophoresis (AFIGE) and clamped homogeneous electric field (CHEF) gel electrophoresis. The rate of DNA dsb rejoining was compared to the rate of rejoining of chromatin breaks measured, also in the same cell population, using the technique of premature chromosome condensation (PCC). Two radiation exposures, 25 Gy and/or 50 Gy, were used and applied to the individual parts of the experiments according to the sensitivity of the assay under investigation. Similar values for the initial rate of DNA dsb rejoining were obtained with all assays used, with t 1/2 ranging between 10 and 12 min after exposure to 25 Gy and between 15 and 20 min after exposure to 50 Gy. The initial rate of rejoining of chromatin breaks was slower than that of DNA dsb and occurred with t 1/2 of 87 min. The results suggest that all major techniques currently used for assaying rejoining of DNA dsb give similar results despite their widely different biophysical basis, and indicate that more information is required before a direct correlation between rejoining of DNA dsb and rejoining of chromatin breaks can be established.
Gabriel E Pantelias - One of the best experts on this subject based on the ideXlab platform.
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comparison of dna double strand break rejoining as measured by pulsed field gel electrophoresis neutral Sucrose Gradient centrifugation and non unwinding filter elution in irradiated plateau phase cho cells
International Journal of Radiation Biology, 1991Co-Authors: George Iliakis, D Blocher, Lorenz Metzger, Gabriel E PanteliasAbstract:SummaryThe initial (up to 30 min) rate of DNA double-strand break (dsb) rejoining was measured in irradiated plateau-phase CHO cells, in a set of parallel experiments using the same cell suspension, by means of non-unwinding filter elution, neutral Sucrose Gradient centrifugation, and two pulsed-field gel electrophoresis assays: asymmetric field inversion gel electrophoresis (AFIGE) and clamped homogeneous electric field (CHEF) gel electrophoresis. The rate of DNA dsb rejoining was compared to the rate of rejoining of chromatin breaks measured, also in the same cell population, using the technique of premature chromosome condensation (PCC). Two radiation exposures, 25 Gy and/or 50 Gy, were used and applied to the individual parts of the experiments according to the sensitivity of the assay under investigation. Similar values for the initial rate of DNA dsb rejoining were obtained with all assays used, with t1/2 ranging between 10 and 12 min after exposure to 25 Gy and between 15 and 20 min after exposure ...
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comparison of dna double strand break rejoining as measured by pulsed field gel electrophoresis neutral Sucrose Gradient centrifugation and non unwinding filter elution in irradiated plateau phase cho cells
International Journal of Radiation Biology, 1991Co-Authors: George Iliakis, D Blocher, Lorenz Metzger, Gabriel E PanteliasAbstract:The initial (up to 30 min) rate of DNA double-strand break (dsb) rejoining was measured in irradiated plateau-phase CHO cells, in a set of parallel experiments using the same cell suspension, by means of non-unwinding filter elution, neutral Sucrose Gradient centrifugation, and two pulsed-field gel electrophoresis assays: asymmetric field inversion gel electrophoresis (AFIGE) and clamped homogeneous electric field (CHEF) gel electrophoresis. The rate of DNA dsb rejoining was compared to the rate of rejoining of chromatin breaks measured, also in the same cell population, using the technique of premature chromosome condensation (PCC). Two radiation exposures, 25 Gy and/or 50 Gy, were used and applied to the individual parts of the experiments according to the sensitivity of the assay under investigation. Similar values for the initial rate of DNA dsb rejoining were obtained with all assays used, with t 1/2 ranging between 10 and 12 min after exposure to 25 Gy and between 15 and 20 min after exposure to 50 Gy. The initial rate of rejoining of chromatin breaks was slower than that of DNA dsb and occurred with t 1/2 of 87 min. The results suggest that all major techniques currently used for assaying rejoining of DNA dsb give similar results despite their widely different biophysical basis, and indicate that more information is required before a direct correlation between rejoining of DNA dsb and rejoining of chromatin breaks can be established.
Paul P Van Veldhoven - One of the best experts on this subject based on the ideXlab platform.
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isolation of plasma membranes and golgi apparatus from a single chicken liver homogenate
Journal of Cellular Biochemistry, 1999Co-Authors: Lieve Vleurick, Eduard Kuhn, Eddy Decuypere, Paul P Van VeldhovenAbstract:Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous Sucrose Gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous Sucrose Gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.
Lieve Vleurick - One of the best experts on this subject based on the ideXlab platform.
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isolation of plasma membranes and golgi apparatus from a single chicken liver homogenate
Journal of Cellular Biochemistry, 1999Co-Authors: Lieve Vleurick, Eduard Kuhn, Eddy Decuypere, Paul P Van VeldhovenAbstract:Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous Sucrose Gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous Sucrose Gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.
Faustino Mollinedo - One of the best experts on this subject based on the ideXlab platform.
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lipid raft isolation by Sucrose Gradient centrifugation and visualization of raft located proteins by fluorescence microscopy the use of combined techniques to assess fas cd95 location in rafts during apoptosis triggering
Methods of Molecular Biology, 2021Co-Authors: Consuelo Gajate, Faustino MollinedoAbstract:Lipid rafts are heterogeneous membrane domains enriched in cholesterol, sphingolipids, and gangliosides that serve as sorting platforms to compartmentalize and modulate signaling pathways. Death receptors and downstream signaling molecules have been reported to be recruited into these raft domains during the triggering of apoptosis. Here, we provide two protocols that support the presence of Fas/CD95 in lipid rafts during apoptosis, involving lipid raft isolation and confocal microscopy techniques. A detailed protocol is provided for the isolation of lipid rafts, by taking advantage of their resistance to Triton X-100 solubilization at 4 °C, followed by subsequent Sucrose Gradient centrifugation and analysis of the protein composition of the different Gradient fractions by Western blotting. In addition, we also provide a detailed protocol for the visualization of the coclustering of Fas/CD95 death receptor and lipid rafts, as assessed by using anti-Fas/CD95 antibodies and fluorescent dye-conjugated cholera toxin B subunit that binds to ganglioside GM1, a main component of lipid rafts, by immunofluorescence and confocal microscopy. These protocols can be extended to any protein of interest to be analyzed for its association to lipid rafts.
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isolation of lipid rafts through discontinuous Sucrose Gradient centrifugation and fas cd95 death receptor localization in raft fractions
Methods of Molecular Biology, 2017Co-Authors: Consuelo Gajate, Faustino MollinedoAbstract:Lipid raft domains, enriched in sphingolipids and cholesterol, serve as sorting platforms and hubs for signal transduction proteins, and show resistance to detergent solubilization. Despite rafts have been involved in survival processes, these membrane domains have also been shown to play a major role in the modulation of death receptor signaling. Here, we describe a detailed protocol for isolating lipid rafts from whole cells by taking advantage of the lipid raft resistance to Triton X-100 solubilization at 4 °C, followed by Sucrose Gradient centrifugation, with subsequent analysis of Fas/CD95 death receptor localization in the raft fractions by immunoblotting. This method is also useful to localize additional proteins in membrane rafts.
