OxiSelect™ Superoxide Dismutase Activity Assay

N° du produit ABIN2344995

Détail du produit

Reactivité: Others

Application: Activity Assay (AcA)

Marque: OxiSelect™

Type d'échantillon: Plasma, Cell Samples, Serum, Tissue Samples

Attributs du produit: The OxiSelect™ Superoxide Dismutase Activity Assay is a fast and reliable kit for the measurement of SOD activity from cell lysate, plasma, serum, tissue homogenates. Each Trial Size Superoxide Dismutase Activity Assay Kit provides sufficient reagents to perform up to 20 assays, including blanks, SOD standards and unknown protein samples.

Ingrédients:

1. SOD Standard : One 20 μL tube provided at 5 Units/μL. Unit Definition: One unit will inhibit the rate of reduction of cytochrome c by 50% in a coupled system, using xanthine and xanthine oxidase, at pH 7.8 at 25°C in a 3.0 ml reaction volume.
2. Xanthine Solution : One 125 μL tube.
3. Xanthine Oxidase Solution, 150X : One 10 μL tube.
4. Chromagen Solution : One 125 μL amber tube.
5. SOD Assay Buffer, 10X : One 1.5 mL tube.

Matériel non inclus:

1. 96-well microtiter plate
2. 37 °C incubator or water bath
3. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
4. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
5. Multichannel micropipette reservoir
6. Microplate reader capable of reading at 490 nm

Information d'application

Indications d'application: Optimal working dilution should be determined by the investigator.

Commentaires:

• Measure superoxide dismutase activity using a standard microplate reader
• Suitable for use with plasma, serum, cells or tissues
• Superoxide dismutase standard included for absolute quantitation

Protocole: Superoxide anions (O2-) are generated by a Xanthine/Xanthine Oxidase (XOD) system, and then detected with a Chromagen Solution. However, in the presence of SOD, these superoxide anion concentrations are reduced, yielding less colorimetric signal.

Préparation des réactifs:

• 1X SOD Assay Buffer: Dilute one vial of 10X SOD Assay Buffer to 1X with deionized water. Mix to homogeneity. Keep the second vial of 10X SOD Assay Buffer undiluted.
• 1X Xanthine Oxidase Solution: Just prior to use, dilute the 150X Xanthine Oxidase Solution to 1X with 1X SOD Assay Buffer. Mix to homogeneity. Special Precautions Avoid the use of reducing agents, such as DTT, in the assay due to interference with the Chromagen Solution.

Préparation de l'échantillon:

• Suspension Cells: Centrifuge 3-6 x 106 cells at 700 x g for 2 minutes and discard supernatant. Wash cell pellet once with ice-cold PBS, centrifuge, and discard the supernatant. Resuspend cell pellet in 0.5 mL of cold 1X Lysis Buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1 mM EDTA). Lyse cells with sonication or homogenation. Centrifuge at 12000 x g for 10 minutes and collect the cell lysate supernatant.
• Adherent Cells: Wash 1-5 x 106 cells once with 10 mL ice-cold PBS per 100 mm dish. Harvest cells with a cell with a cell scraper in 1 mL of cold 1X Lysis Buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1 mM EDTA). Lyse cells with sonication or homogenation. Centrifuge at 12000 x g for 10 minutes and collect the cell lysate supernatant.
• Tissue Lysates: Homogenize tissue sample in 5-10 mL of cold 1X Lysis Buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1 mM EDTA) per gram tissue. Lyse cells with sonication or homogenation. Centrifuge at 12000 x g for 10 minutes and collect the tissue lysate supernatant.
• Plasma: Collect blood with an anticoagulant such as heparin, citrate or EDTA and mix by inversion. Centrifuge the blood at 1000 x g at 4 °C for 10 minutes. Collect plasma supernatant without disturbing the white buffy layer. Sample should be tested immediately or frozen at -80 °C for storage.
• Serum: Collect blood in a tube with no anticoagulant. Allow the blood to clot at room temperature for 30 minutes. Centrifuge at 2500 x g for 20 minutes. Remove the yellow serum supernatant without disturbing the white buffy layer. Samples should be tested immediately or frozen at -80 °C for storage. 4

Procédure de l'essai:

1. Prepare samples including a blank in a 96-well microtiter plate according to the below table. Allow pre-incubation time if inhibitor is used. Component Blank Sample SOD Sample 0 μL X μL Inhibitor (optional) 0 μL Y μL Xanthine Solution 5 μL 5 μL Chromagen Solution 5 μL 5 μL 10X SOD Assay Buffer 10 μL 10 μL DI Water 70 μL 70-(X+Y) μL Total 90 μL 90 μL
2. Finally, add 10 μL of pre-diluted 1X Xanthine Oxidase Solution (see Preparation of Reagents) to each well. Mix well and incubate for 1 hour at 37 °C.
3. Read absorbance at 490 nm on a microplate reader. Preparation of SOD Standards (Optional)
   1. Thaw SOD Standard at 4 °C.
   2. Freshly prepare a dilution series (1:4 is suggested) of SOD Standard in the concentration range of 5 Units/μL - 1.2 mU/μL by diluting the SOD Standard in 1X Assay Buffer (see Preparation of Reagents).
   3. Transfer 10 μL of each dilution to a 96-well microtiter plate, including a 1X Assay Buffer blank.
   4. Prepare the following master mixture, adjusting for the required number of wells. Component Volume per well Xanthine Solution 5 μL Chromagen Solution 5 μL 10X SOD Assay Buffer 10 μL DI Water 60 μL Total 80 μL
   5. Transfer 80 μL of the above master mixture to each well. 5
   6. Finally, add 10 μL of pre-diluted 1X Xanthine Oxidase Solution (see Preparation of Reagents) to each well. Mix well and incubate for 1 hour at 37 °C.
   7. Read absorbance at 490 nm on a microplate reader.

Restrictions: For Research Use only

Stockage

Stock: -20 °C

Stockage commentaire: Store kit components at -20°C. Avoid multiple freeze/thaws by aliquoting. The Chromagen Solution is light sensitive and should be maintained in amber tubes.

Référence

Kim, Lee, Cheon, Nam, Oh, Kim: "Antioxidant and hepatoprotective effects of fermented red ginseng against high fat diet-induced hyperlipidemia in rats." dans: Laboratory animal research, Vol. 32, Issue 4, pp. 217-223, (2017) (PubMed).

Abdallah, Farag, Osman, Kim, Kang, Pan, Abdel-Sattar: "Isolation of major phenolics from Launaea spinosa and their protective effect on HepG2 cells damaged with t-BHP." dans: Pharmaceutical biology, Vol. 54, Issue 3, pp. 536-41, (2016) (PubMed).

Murat, Korhan, Kizer, Evcim, Kefi, Demir, Gidener, Atabey, Esen: "Resveratrol Protects and Restores Endothelium-Dependent Relaxation in Hypercholesterolemic Rabbit Corpus Cavernosum." dans: The journal of sexual medicine, Vol. 13, Issue 1, pp. 12-21, (2016) (PubMed).

Song, Seo, Kim, Moon, Won, Son, Son, Kwon: "Protective Effects of Manassantin A against Ethanol-Induced Gastric Injury in Rats." dans: Biological & pharmaceutical bulletin, Vol. 39, Issue 2, pp. 221-9, (2016) (PubMed).

Liu, Shen, He, Chang, Shi, Tao, Li, Xun, Guo, Yu, Wang: "Noise induced hearing loss impairs spatial learning/memory and hippocampal neurogenesis in mice." dans: Scientific reports, Vol. 6, pp. 20374, (2016) (PubMed).

Erbaş, Yılmaz, Taşkıran: "Levetiracetam attenuates rotenone-induced toxicity: A rat model of Parkinson's disease." dans: Environmental toxicology and pharmacology, Vol. 42, pp. 226-30, (2016) (PubMed).

Bozkurt, Ege, Aysul, Akşit, Tüzün, Küçükyılmaz, Borum, Uygun, Akşit, Aypak, Şimşek, Seyrek, Koçer, Bintaş, Orojpour: "Effect of anticoccidial monensin with oregano essential oil on broilers experimentally challenged with mixed Eimeria spp." dans: Poultry science, Vol. 95, Issue 8, pp. 1858-68, (2016) (PubMed).

Kara, Sari, Akşit, Yay, Akşit, Dönmez: "Effects of selenium on ischaemia-reperfusion injury in a rat testis model." dans: Andrologia, Vol. 48, Issue 10, pp. 1267-1273, (2016) (PubMed).

Bozkurt, Bintaş, Kırkan, Akşit, Küçükyılmaz, Erbaş, Çabuk, Akşit, Parın, Ege, Koçer, Seyrek, Tüzün: "Comparative evaluation of dietary supplementation with mannan oligosaccharide and oregano essential oil in forced molted and fully fed laying hens between 82 and 106 weeks of age." dans: Poultry science, Vol. 95, Issue 11, pp. 2576-2591, (2016) (PubMed).

Girgih, Alashi, He, Malomo, Raj, Netticadan, Aluko: "A novel hemp seed meal protein hydrolysate reduces oxidative stress factors in spontaneously hypertensive rats." dans: Nutrients, Vol. 6, Issue 12, pp. 5652-66, (2015) (PubMed).

Akgüllü, Hekim, Ery?lmaz, Boyac?o?lu, Güngör, Meteo?lu, Karul, Onba??l?: "The usefulness of carvedilol and nebivolol in preventing contrast nephropathy in rats." dans: Renal failure, Vol. 37, Issue 3, pp. 511-7, (2015) (PubMed).

Kim, Kang, Nam, Cho, Chung, Kim, Kim, Cho, Hong, Sohn, Shin: "Ameliorating the effect of astragaloside IV on learning and memory deficit after chronic cerebral hypoperfusion in rats." dans: Molecules (Basel, Switzerland), Vol. 20, Issue 2, pp. 1904-21, (2015) (PubMed).

Rajapaksha, Stanley, Todd: "Effects of macromolecular crowding on the structure of a protein complex: a small-angle scattering study of superoxide dismutase." dans: Biophysical journal, Vol. 108, Issue 4, pp. 967-74, (2015) (PubMed).

Medlow, McEneny, Murphy, Trinick, Duly, Davison: "Exercise training protects the LDL I subfraction from oxidation susceptibility in an aged human population." dans: Atherosclerosis, Vol. 239, Issue 2, pp. 516-22, (2015) (PubMed).

Wang, Cao, Chai, Liao, Huang, Tang: "Oxidative damage of naphthenic acids on the Eisenia fetida earthworm." dans: Environmental toxicology, (2015) (PubMed).

Cheng, Kao, Ma, Hung, Wang, Liu, Chen, Tsai: "SIRT1-related inhibition of pro-inflammatory responses and oxidative stress are involved in the mechanism of nonspecific low back pain relief after exercise through modulation of Toll-like receptor 4." dans: Journal of biochemistry, Vol. 158, Issue 4, pp. 299-308, (2015) (PubMed).

Lee, Yang, Huang, Li, Huang, Kao, Chen, Chen: "Exercise suppresses COX-2 pro-inflammatory pathway in vestibular migraine." dans: Brain research bulletin, Vol. 116, pp. 98-105, (2015) (PubMed).

Tarhan, Özdemir, ?ncesu, Demirkan: "Direct and protective effects of single or combined addition of vincristine and ?-viniferin on human HepG2 cellular oxidative stress markers in vitro." dans: Cytotechnology, (2015) (PubMed).

Hunter, Hosgood, Barlow, Nicholson: "Ischaemic conditioning reduces kidney injury in an experimental large-animal model of warm renal ischaemia." dans: The British journal of surgery, Vol. 102, Issue 12, pp. 1517-25, (2015) (PubMed).

Pérez, Olmo, Teijón, Muñíz, Montero, Teijón, Blanco: "Biocompatibility evaluation of pH and glutathione-responsive nanohydrogels after intravenous administration." dans: Colloids and surfaces. B, Biointerfaces, Vol. 136, pp. 222-31, (2015) (PubMed).

Détail du antigène

Sujet: Reactive oxygen species (ROS), such as superoxide (O -2 ) and hydrogen peroxide (H2O2), are constantly produced during metabolic processes in all living species. Under normal physiological conditions, cellular ROS generation is counterbalanced by the action of antioxidant enzymes and other redox molecules. However, excessive ROS accumulation will lead to cellular injury, such as damage to DNA, protein, and lipid membrane. Because of their potential harmful effects, excessive ROS must be promptly eliminated from the cells by a variety of antioxidant defense mechanisms. Superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen, is one of the most important antioxidative enzymes. SOD enzymes are classified into three groups: cytosolic Cu/Zn-SOD, mitochondrial Mn-SOD, and extracellular Ec-SOD. Our OxiSelect™ Superoxide Dismutase Activity Assay uses a xanthine/xanthine oxidase (XOD) system to generate superoxide anions. The included chromagen produces a water-soluble formazan dye upon reduction by superoxide anions. The activity of SOD is determined as the inhibition of chromagen reduction (See Figure 1).