Dissertation

Antioxidant Enzyme Assay Protocol

MEASUREMENT OF SUPEROXIDE DISMUTASE, CATALASE, AND ... MEASUREMENT OF SUPEROXIDE DISMUTASE, CATALASE, AND ...
Cells contain a large number of antioxidants to prevent or repair the damage caused by ROS, as well as to regulate redox-sensitive signaling pathways General protocols are described to…

Antioxidant Enzyme Assay Protocol

Add 1 ml naoh (5n) dropwise with a 5 glass pasteur pipette and 2 ml bulb and mix until dissolved. The heparin-binding domain is important because it mediates the binding of ecsod to cells. Prepare immediately before use and allow a 1 h waiting period for the xo to stabilize its activity.

This recipe is for a 5 acrylamide stacking gel (wtvol) and is used with all native gels. While the amount of sod in total will give you a good idea of the overall superoxide detoxifying capacity of a cell or tissue, determination of both total sod and mnsod, and subsequently cuznsod, will be more informative as location and responsiveness of mnsod and cuznsod differ. The adenoviral vector ad mcf 10a cells transduced with increasing moi of adenoviral catalase (adcatalase) were subjected to native gel electrophoresis (100 g proteinwell) and stained as described (a).

Inhibition of experimental pulmonary metastasis by controlling biodistribution of catalase in mice. Temed (28 mm) and 8 l of stock riboflavin-5-phosphate (0. Redox regulation of pancreatic cancer cell growth role of glutathione peroxidase in the suppression of the malignant phenotype.

This standard is utilized only to ensure that the assay is working correctly. Gold-conjugated goat anti-rabbit igg (ge healthcare life sciences, cat. Both of the catalase assays should be preformed utilizing a positive control.

Make sure to add blocking solution only as a negative control to separate wells. Wong hwg, elwell jh, oberley lw, goeddel dv. Heterologous expression of selenium-dependent glutathione peroxidase afford cellular resistance to paraquat.

Enzymes play an important role in lowering the ros levels and helping avoid oxidative stress. Add dapi to a final dilution of 15000 in the same tube as secondary antibody add 300 l to each well of cells. Mix all reagents together in a 50 ml conical tube. To determine a change in activity of the antioxidant proteins cuznsod and mnsod in mcf-10a immortalized breast epithelial cells after infection with the ad (50 or 100 multiplicity of infectivity, moi) adenoviral vector constructs, an activity gel was performed. Pause point slides can be store for 2 mo or more at 4c to help retain fluorescent signal.


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Antioxidant Enzyme Assay Protocol

Protocol (Step-by-step) for Antioxidant Enzyme Assay?
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Antioxidant Enzyme Assay Protocol For negative control, add only blocking buffer with no antibody (300 l). An assay for superoxide dismutase in mammalian tissue homogenates. Make fresh each time of use and store at room temperature. Image the gels on a computer flat bed scanner with light from top and bottom or utilize other available imaging systems. Also, ecsod is a glycosylated high molecular weight homotetramer (155 kda), while cuznsod is an unglycosylated homodimer (32 kda). Localization of cuznsod protein expression is visualized following ad transduction (300 moi) as robust red staining throughout the cytoplasm (1100 mnsod 1ab cy2 goat-anti-rabbit 2ab) and the nuclei appear blue following dapi counterstain (b). Thus, Sonicator (equipped with a cuphorn, model vs 750, sonics and materials, inc. Mean gray-level pixel values were then obtained (a). Studies on the quantitation and qualitative characterization of erythrocyte glutathione peroxidase. The role of manganese superoxide dismutase in the growth of pancreatic adenocarcinoma. Highly specific immunostains for both tissues and cells are available for sod, gpx, and catalase.
  • Spectrophotometric Assays for Antioxidant Enzymes in ...


    Labeling of mnsod was extremely light in cells treated with the ad demonstrated increases in labeling (arrow) in the mitochondria (bottom panel) (b). Catalase also attenuates both the basal and mnsod-dependent expression of mmps and collagen deposition cytosolic glutathione peroxidase (gpx, gpx1) is a selenoprotein, first described as an enzyme that protects hemoglobin from oxidative degradation in red blood cells , gpx requires several secondary enzymes (glutathione reductase and glucose-6-phosphate dehydrogenase) and cofactors (reduced glutathione, nadph, and glucose 6-phosphate) to function at high efficiency. Catalase gels (8 gels) will have one band (220 kda) that rarely saturates getting larger with increasing catalase activity. Leave under light, and on the light box for 15 min to 2 h making sure the gel does not dry out. Vibra cell cup horn sonicator at 40 power.

    Make sure to cover the cells with aluminum foil to protect the secondary antibody from light. For the catalase activity result to be accurate, the resultant absorbance plot should display an exponential decay curve for at least 1 min of the 2 min recording time. Heterologous expression of selenium-dependent glutathione peroxidase afford cellular resistance to paraquat. Localization of cuznsod protein expression is visualized following ad transduction (300 moi) as robust red staining throughout the cytoplasm (1100 mnsod 1ab cy2 goat-anti-rabbit 2ab) and the nuclei appear blue following dapi counterstain (b). Eppendorf tube and sonicate for 1 min in 30 sec bursts (with cooling on ice in between) using a vibra cell cup horn sonicator at 40 power.

    Recently, there has been some evidence showing the presence of cuznsod (approximately 2) in the intermembrane space of mitochondria and this localization was suggested to be important in providing further protection against ros and in preventing superoxide radicals from leaking out of the mitochondria. The sods convert superoxide radical into hydrogen peroxide and molecular oxygen (o ), while the catalase and peroxidases convert hydrogen peroxide into water and in the case of catalase to oxygen and water. Western blots, activity gels and activity assays are various methods used to determine protein and activity in both cells and tissue depending on the amount of protein needed for each assay. For sod stained gels, run duplicate samples on two 12 (volvol) native gels. Furthermore, an additional stop-flow kinetics assay cannot accurately determine sod within cell and tissue homogenates is also based on the inhibition of the reduction of nbt by sod originally described by ornstein to interact with nbt reducing the yellow tetrazolium within the gel to a blue precipitate. Prepare the assay solutions and add to tubes in the order shown in the tables below. Human copper-containing superoxide dismutase of high molecular weight. Bsa (1, wtvol), 10 l of triton x (1, volvol), 5 l of tween-20 (0. Adjust the volume to 300 ml with ddh acrylamide and bis-acrylamide are toxic. Thus, the assay using cumene hydroperoxide or tert-butyl hydroperoxide measures selenium-dependent gpx and activity from glutathione s-transferases (selenium-independent gpx).

    Antioxidant enzyme responses to moderate water ... Cite this protocol as: Elavarthi S., Martin B. (2010) Spectrophotometric Assays for Antioxidant Enzymes in ...

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