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[1.] Sj/Fragment 031 10 - Diskussion
Bearbeitet: 28. November 2016, 22:59 (Hindemith)
Erstellt: 26. November 2016, 23:53 LieschenMueller
Fragment, KeineWertung, Palmada et al 2006, SMWFragment, Schutzlevel, Sj, Unfertig

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LieschenMueller
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Untersuchte Arbeit:
Seite: 31, Zeilen: 10
Quelle: Palmada et al 2006
Seite(n): 422, Zeilen: r.col:18f
Tritium-labeled 2-deoxy-D-glucose (2-DOG) was used as the glucose analogue for uptake determination. Groups of 10-15 oocytes injected with GLUT4 were incubated for 2 h with 1 μM insulin. Thereafter oocytes were placed in 0.25 ml of ND96 (96 mmol/l NaCl, 2 mmol/l KCl, 1.8 mmol/l CaCl2, 1 mmol/l MgCl2 and 5 mmol/l HEPES, pH 7.4) containing 37 KBq 3H 2-DOG and 50 μmol/l (or the indicated amount for kinetic analysis) of unlabeled 2-DOG. After incubation for 10 min at room temperature with 2-DOG (linear range of uptake), uptake was terminated by washing the oocytes four times with 3 ml of ice-cold phosphate- buffered saline (PBS) containing 100 mmol/l 2-DOG. Oocytes were individually transferred into scintillation vials and dissolved by adding 200 μl of 10% SDS before radioactivity was determined.

In HEK-293 cells, 2-DOG uptake was measured 2 days after transfection by incubating cells at 37°C for 5 min (linear range of uptake) in glucose free krebs Ringer HEPES buffer containing 3H 2-DOG 0.1 μCi/well and 0.3 mmol/l cold 2-DOG with or without 0.1 mmol/l phloretein. Uptake was terminated by rapid aspiration of uptake solution and washing four times with ice-cold PBS containing 50 mmol/l unlabeled 2-DOG. Thereafter, cells were lyses with 10mmol/l NaOH/0.1%/Triton-X-100, and radioactivity incorporated into the cells was measured with a liquid scintillation counter. Protein concentrations were determined by the Bradford method. For kinetic analyis, 2-DOG uptake was measured by incubating cells with 3H 2-DOG 0.1 μCi/well and various concentrations of unlabeled 2-DOG at 37°C for 5 minutes.

In isolated adipocytes, 2-DOG uptake was measured by incubating the cells with 0.1 μCi 3H 2-DOG and 0.1 mmol/l cold 2-DOG with or without 0.1 mmol/l phloretin.

Transport studies. Tritium-labeled 2-deoxy-D-glucose (2-DOG) was used as the glucose analog for uptake determination. In Xenopus oocytes, the transport assay was performed 4 days after cRNA injection and contained 10–15 single oocytes in 0.25 ml ND96 (96 mmol/l NaCl, 2 mmol/l KCl, 1.8 mmol/l CaCl2, 1 mmol/l MgCl2, and 5 mmol/l HEPES, pH 7.4) containing 1 μCi 3H 2-DOG and 50 μmol/l (or the indicated amount in the kinetic analysis) of unlabeled 2-DOG. After incubation for 30 min at room temperature with

Page 423

2-DOG (linear range of uptake), uptake was terminated by washing the oocytes four times with 3 ml ice-cold PBS containing 100 mmol/l unlabeled 2-DOG. Oocytes were individually transferred into scintillation vials and dissolved by adding 200 μl of 10% SDS before the radioactivity was determined. In HEK-293 cells, 2-DOG uptake was measured 2 days after transfection by incubating the cells at 37°C for 5 min (linear range of uptake) in glucose-free Krebs Ringer HEPES buffer containing 3H 2-DOG 0.1 μCi/well and 0.3 mmol/l cold 2-DOG with or without 0.1 mmol/l phloretin. Uptake was terminated by rapid aspiration of uptake solution and washing four times with ice-cold PBS containing 50 mmol/l unlabeled 2-DOG. Thereafter, cells were lysed with 10 mmol/l NaOH/0.1% Triton X-100, and radioactivity incorporated into the cells was measured with a liquid scintillation counter. Protein concentrations were determined by the Bradford method. For kinetic analysis, 2-DOG uptake was measured by incubating the cells with 3H 2-DOG 0.1 Ci/well and various concentrations of unlabeled 2-DOG at 37°C for 5 min. In isolated adipocytes, 2-DOG uptake was measured by incubating the cells with 0.1 μCi 3H 2-DOG and 0.1 mmol/l cold 2-DOG with or without 0.1 mmol/l phloretin.

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