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Western Blot Analysis Cytosol proteins from lung (50 ug) and liver (10 g) were separated on a 12% (w/v) polyacrylamide gel in an electrophoresis system (Novex, San Diego, CA) . After running at 200 V, the protein bands were transferred overnight at 40 V onto a nitrocellulose membrane. All membranes were blocked in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween-20) containing 5% (w/v) dry milk for 1 h on a shaker at room temperature. Membranes were incubated with either rabbit anti-AST IV or rabbit anti- STa (1:5000) in TBST containing 5% (w/v) dry milk for 2 h on a shaker at room temperature. After incubation, membranes were washed with TBST for 4 × 15 min and incubated with secondary antibody (horseradish peroxidase-conjugated immuno-pure goat anti-rabbit IgG; H+L) at 1:5000 dilutions in the same buffer for 2 h. The membranes were washed with TBST for 4× 15 min and then with Tris buffered saline (TBS) 3 × 5 min. Fluorescent bands were developed with 1mL of substrate containing same volume of each Super Signal West Pico Luminol Enhancer solution and Super Signal West Pico Stable Peroxidase solution at room temperature for 5 min. The X-ray films were exposed to the membrane and then developed. Films were scanned and the densitometry analysis was performed with AAB software in a Gel Documentation and Analysis System from Advanced American Biotechnology Determination of Nonprotein Soluble Thiol(NPSH) Total nonprotein soluble thiol in lung and liver cytosol was determined by the standard DTNB (Ellman’s reagent, 5,5`-dithiobis-2-nitrobenzoic acid) method with a slight modification. Lung and liver tissues were homogenized with 50 mM Tris buffer, pH 7.5, containing 250 mM sucrose and 5 mM EDTA. Homogenates were centrifuged at 100,000g for 1 h. Equal volume of 5% (w/v) sulfosalisialic acid was used to precipitate all proteins from the tissue cytosols. After mild vortexing and centrifugation at 4000g, 80 uL of transparent sample supernatant was added to 720 uL of 0.1 M potassium phosphate buffer containing 5 uM DTNB. The absorbance was measured after 5 min at 412 nm in a UV-spectrophotometer. The steady state of the reaction kinetics was checked up to 7 min. A standard curve was generated using GSH and individual sample values were determined from this standard curve |
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yinjuanchen
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It seems very easy try to translate by yourself. I believe Iyou can do it very well.
2楼2011-05-21 15:15:45
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