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Glutathione Colorimetric Assay Kit Kit

D Reactivity: Chemical Colorimetric Cell Lysate, Plasma, Serum, Tissue Samples
Pubmed (20)
Catalog No. ABIN411672
$425.00
Plus shipping costs $45.00
100 tests
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Delivery in 2 to 3 Business Days
  • Target
    Glutathione
    Reactivity
    • 8
    • 4
    • 3
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Chemical
    Detection Method
    Colorimetric
    Application
    Detection (D)
    Brand
    ApoGSH™
    Sample Type
    Cell Lysate, Plasma, Serum, Tissue Samples
    Specificity
    ApoGSH TM Glutathione Colorimetric Assay Kit provides a convenient, colorimetric method for analyzing either total glutathione or the reduced form glutathione alone using a microtiter plate reader. The assay is based on the glutathione recycling system by DTNB and glutathione reductase (fig. 1). DTNB and glutathione (GSH) react to generate 2-nitro-5-thiobenzoic acid which has yellow color. Therefore, GSH concentration can be determined by measuring absorbance at 412 nm. The generated GSSG can be reduced back to GSH by glutathione reductase, and GSH reacts with DTNB again to produce more 2-nitro-5-thiobenzoic acid. Therefore, the recycling system dramatically improves the sensitivity of total glutathione detection. The kit includes the 5-Sulfosalicylic acid (SSA) for the removal of proteins from samples and for the protection of GSH oxidation and ?-glutamyl transpeptidase reaction. The kit can quantify glutathione from 1- 100 ng/well in a 200 μL reaction. For detecting lower glutathione concentrations, such as in blood samples, increasing reaction time will generate stronger signal. The kit can also specifically detect the reduced form of glutathione (GSH) by omitting the glutathione reductase from the reaction mixture. The sensitivity for detecting the reduced form of glutathione (without recycling system) is 100 times lower than detecting the total glutahione.
    Characteristics
    ApoGSHTM Glutathione Colorimetric Detection Kit: Convenient & Sensitive Colorimetric Assay for analyzing either Total Glutathione or Reduced form of Glutathione from Tissues, Cells, Plasma & Erythrocytes.
    Components
    Glutathione Reaction Buffer
    Glutathione Substrate (DTNB)
    NADPH Generating Mix (lyophilized)
    Glutathione Reductase (lyophilized)
    Sulfosalicylic Acid (SSA, 1 gram)
    GSH Standard (lyophilized, MW 307)
  • Application Notes
    The Colorimetric Glutathione Detection Kit provides a simple in vitro assay for detecting the GSH changes in apoptosis and other pathological processes.
    Comment

    Further details regarding sample type: Cell and tissue lysates, culture media, urine, plasma and serum, as well as many other biological fluids

    Protocol
    1. Prepare enough Reaction Mix for the standard and samples to be assayed in 96-well plate (not provided). Each well should contain: 20 µL NADPH Generating Mix 20 µL Glutathione Reductase* 120 µL Glutathione Reaction Buffer * For detecting the reduced form of glutathione only, omit Glutathione Reductase. Use 20 µL of the Glutathione Reaction Buffer replace the 20 µL of glutahione Reductase.
    2. Mix well. Add 160 µL of the Reaction Mix to each well and incubate at room temperature for 10 minutes to generate NADPH.
    3. Add 20 µL of either the GSH standard solutions or the sample solution. Incubate the plate at room temperature for 5-10 min. Note: We recommend to make several dilutions of your sample using the 1 % SSA to make sure the readings are within the range of the standard calibration curve.
    4. Add 20 µL of Substrate solution, and incubate at room temperature for 5-10 min, or longer if the samples contain low levels of glutathione. Notes: a) Since the reaction starts immediately after the addition of substrate, use a multichannel pipette or repeating pipette is recommended to avoid the reaction time lag among wells. b) You can read samples immediately and at various times following addition of the substrate solution for kinetic studies. Read the absorbance at 405 nm or 415 nm using a microplate reader.
    5. Determine concentrations of GSH in the sample solutions using the standard glutathione calibration curve. Note: A. Using reduced form glutathione Standard Curve for detecting reduced form of glutathione. Using total Glutathione Standard Curve for detecting total glutathione. There are about 10 to 100 fold difference in detection sensitivity between detecting reduced form glutathione and total glutathione (see procedure step IV for preparation of standard curve). B. The colorimetric reaction is stable and the O.D. increases linearly over 30 min for total glutathione detection.
    Calculation of Results

    Calculation of Total Glutathione Pseudo-end point method: Total Glutathione = (O.D. sample - O.D. blank ) Kinetic method: Total Glutathione = (Slope sample -Slope blank )/Slope VI. Reagent Interference Reducing agents such as ascorbic acid, beta-mercaptoethanol, dithiothreitol (DTT) and cysteine, or thiol reactive compounds such as maleimide compounds, interfere with the glutathione assay and therefore should be avoided during the sample preparation. When detecting the reduced form of glutathione, protein thiols can generate significant background signal. In such cases, it is necessary to completely remove proteins from samples. We suggest using Amicon Centrifugal Spin column with 5K molecular weight cut off filter to remove proteins. Then the reduced glutathione can be easily detected from spin through samples. fig.2. Glutathione Standard Curve. Various amounts of standard glutathione was added to the glutathione reaction and incubated for 10 min according to the kit instructions. Absorbance was measured at O.D. 405 nm. VII.

    Restrictions
    For Research Use only
  • Storage
    -20 °C
    Expiry Date
    12 months
  • Chen, Xu, Zhang, Wang, Wang, Edin, Zeldin, Wang: "CYP2J2 overexpression attenuates nonalcoholic fatty liver disease induced by high-fat diet in mice." in: American journal of physiology. Endocrinology and metabolism, Vol. 308, Issue 2, pp. E97-E110, 2015 (PubMed).

    Kim, Devalaraja-Narashimha, Padanilam: "TIGAR regulates glycolysis in ischemic kidney proximal tubules." in: American journal of physiology. Renal physiology, Vol. 308, Issue 4, pp. F298-308, 2015 (PubMed).

    Cheung, Kwok, To, Lau: "Anti-Atherogenic Effect of Hydrogen Sulfide by Over-Expression of Cystathionine Gamma-Lyase (CSE) Gene." in: PLoS ONE, Vol. 9, Issue 11, pp. e113038, 2014 (PubMed).

    Liu, Palanivel, Rai, Park, Gabor, Scheid, Xu, Sweeney: "Adiponectin stimulates autophagy and reduces oxidative stress to enhance insulin sensitivity during high-fat diet feeding in mice." in: Diabetes, Vol. 64, Issue 1, pp. 36-48, 2014 (PubMed).

    Ye, Fan, Venneti, Wan, Pawel, Zhang, Finley, Lu, Lindsten, Cross, Qing, Liu, Simon, Rabinowitz, Thompson: "Serine catabolism regulates mitochondrial redox control during hypoxia." in: Cancer discovery, Vol. 4, Issue 12, pp. 1406-17, 2014 (PubMed).

    Dinnen, Mao, Qiu, Cassai, Slavkovich, Nichols, Su, Brandt-Rauf, Fine: "Redirecting apoptosis to aponecrosis induces selective cytotoxicity to pancreatic cancer cells through increased ROS, decline in ATP levels, and VDAC." in: Molecular cancer therapeutics, Vol. 12, Issue 12, pp. 2792-803, 2013 (PubMed).

    Srivastava, Rahman, Kashyap, Singh, Jain, Jahan, Lohani, Lantow, Pant: "Nano-titanium dioxide induces genotoxicity and apoptosis in human lung cancer cell line, A549." in: Human & experimental toxicology, Vol. 32, Issue 2, pp. 153-66, 2013 (PubMed).

    Srivastava, Pant, Kashyap, Kumar, Lohani, Jonas, Rahman: "Multi-walled carbon nanotubes induce oxidative stress and apoptosis in human lung cancer cell line-A549." in: Nanotoxicology, Vol. 5, pp. 195-207, 2011 (PubMed).

    Seidel, Roth, Ge, Merfort, Sng, Ammit: "IκBα glutathionylation and reduced histone H3 phosphorylation inhibit eotaxin and RANTES." in: The European respiratory journal, Vol. 38, Issue 6, pp. 1444-52, 2011 (PubMed).

    Zhang, Lau, Monks: "The cytoprotective effect of N-acetyl-L-cysteine against ROS-induced cytotoxicity is independent of its ability to enhance glutathione synthesis." in: Toxicological sciences : an official journal of the Society of Toxicology, Vol. 120, Issue 1, pp. 87-97, 2011 (PubMed).

    Henderson, Li, He, Zhang, Malfatti, Gandara, Grimminger, Danenberg, Beckett, de Vere White, Turteltaub, Pan: "A microdosing approach for characterizing formation and repair of carboplatin-DNA monoadducts and chemoresistance." in: International journal of cancer. Journal international du cancer, 2010 (PubMed).

    Howard, Tahir, Javed, Waring, Ford, Hirst: "Glycine transporter GLYT1 is essential for glycine-mediated protection of human intestinal epithelial cells against oxidative damage." in: The Journal of physiology, Vol. 588, Issue Pt 6, pp. 995-1009, 2010 (PubMed).

    Jeannot, Mellottee, Bioulac-Sage, Balabaud, Scoazec, Tran Van Nhieu, Bacq, Michalak, Buob, Laurent-Puig, Rusyn, Zucman-Rossi: "Spectrum of HNF1A somatic mutations in hepatocellular adenoma differs from that in patients with MODY3 and suggests genotoxic damage." in: Diabetes, Vol. 59, Issue 7, pp. 1836-44, 2010 (PubMed).

    Stamper, Bammler, Beyer, Farin, Nelson: "Differential regulation of mitogen-activated protein kinase pathways by acetaminophen and its nonhepatotoxic regioisomer 3'-hydroxyacetanilide in TAMH cells." in: Toxicological sciences : an official journal of the Society of Toxicology, Vol. 116, Issue 1, pp. 164-73, 2010 (PubMed).

    Macias-Gonzalez, Cardona, Queipo-Ortuño, Bernal, Martin, Tinahones: "PPARgamma mRNA expression is reduced in peripheral blood mononuclear cells after fat overload in patients with metabolic syndrome." in: The Journal of nutrition, Vol. 138, Issue 5, pp. 903-7, 2008 (PubMed).

    Nefedova, Fishman, Sherman, Wang, Beg, Gabrilovich: "Mechanism of all-trans retinoic acid effect on tumor-associated myeloid-derived suppressor cells." in: Cancer research, Vol. 67, Issue 22, pp. 11021-8, 2007 (PubMed).

    Karuri, Huang, Bodreddigari, Sutter, Roebuck, Kensler, Sutter: "3H-1,2-dithiole-3-thione targets nuclear factor kappaB to block expression of inducible nitric-oxide synthase, prevents hypotension, and improves survival in endotoxemic rats." in: The Journal of pharmacology and experimental therapeutics, Vol. 317, Issue 1, pp. 61-7, 2006 (PubMed).

    Mutlu, Snyder, Bellmeyer, Wang, Hawkins, Soberanes, Welch, Ghio, Chandel, Kamp, Sznajder, Budinger: "Airborne particulate matter inhibits alveolar fluid reabsorption in mice via oxidant generation." in: American journal of respiratory cell and molecular biology, Vol. 34, Issue 6, pp. 670-6, 2006 (PubMed).

    Powell, Kosyk, Ross, Schoonhoven, Boysen, Swenberg, Heinloth, Boorman, Cunningham, Paules, Rusyn: "Phenotypic anchoring of acetaminophen-induced oxidative stress with gene expression profiles in rat liver." in: Toxicological sciences : an official journal of the Society of Toxicology, Vol. 93, Issue 1, pp. 213-22, 2006 (PubMed).

    Huang, Dai, Barbacioru, Sadée: "Cystine-glutamate transporter SLC7A11 in cancer chemosensitivity and chemoresistance." in: Cancer research, Vol. 65, Issue 16, pp. 7446-54, 2005 (PubMed).

  • Target
    Glutathione
    Synonyms
    GT, ILBP, ILLBP, PIP, I-15P, I-BABP, ILBP3, Illbp, I-BALB, I-BAP, fatty acid binding protein 6, fatty acid binding protein 6, ileal (gastrotropin), FABP6, Fabp6
    Target Type
    Chemical
    Background
    Glutathione (GSH) is the major intracellular low-molecular-weight thiol that plays a critical role in the cellular defense against oxidative stress in mammalian cells.
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