Glutathione (GSH,GSSG,Total) Fluorometric Assay Kit

Details for Product No. ABIN411673
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Target Name (Antigen)
Epitope
Whole Molecule
Reactivity
Amino Acid
(1), (2), (1), (1)
Application
Immunoassay (IA)
Pubmed 4 references available
Quantity 100 tests
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Catalog No. ABIN411673
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Sample Type Cell Samples, Tissue Lysate, Cell Culture Supernatant, Urine, Plasma, Serum, Biological Fluids
Detection Method Fluorometric
Specificity The GST Colorimetric Activity Assay Kit is based upon the GST-catalyzed reaction between GSH and the GST substrate, CDNB (1-chloro-2,4-dinitrobenzene, which has the broadest range of isozyme detectability (e.g., alpha-, mu-, pi-, and other GST isoforms). Under certain conditions, the interaction between glutathione and CDNB is totally dependent on the presence of active GST. The GST-catalyzed formation of GS-DNB produces a dinitrophenyl thioether which can be detected by spectrophotometer at 340 nm. One unit of GST activity is defined as the amount of enzyme producing 1 µM of GS-DNB conjugate/min under the conditions of the assay. The kit can detect GST activity in crude cell lysate or purified protein fraction, and also quantitate GST-tagged fusion protein. Detect limit: Active GST 10 ng/assay.
Characteristics Glutathione Assay Kit (GSH,GSSG and Total): Convenient & Simple Fluorometric Assay to Detect GSH, GSSG, and total glutathione from Cells, Tissues, Serum or other Liquid Samples.
Components Glutathione Assay Buffer
PCA (Perchloric Acid6N)
KOH (<6N)
OPA Probe (o-phthalaldehyde )
Reducing Agent Mix
GSH Quencher
GSH Standard (FW: 307)
Target Type Amino Acid
Background Glutathione is the major intracellular low-molecular-weight thiol that plays a critical role in cellular defense against oxidative stress in tissues and cells. Commercially available glutathione detection kits, such as the DTNB-enzyme cycling glutathione assay kit or the Monochlorobimane based assay kit hardly distinguish between reduced glutathione (GSH, FW: 307) and oxidized glutathione (GSSG, FW: 612). Glutathione Detection Kit provides a unique, convenient tool for detecting GSH, GSSG, and total glutathione individually. In the assay, OPA, reacts with GSH (not GSSG), generating fluorescence, so GSH can be specifically quantified. Adding a reducing agent converts GSSG to GSH, so (GSH + GSSG) can be determined. To measure GSSG specifically, a GSH Quencher is added to remove GSH, preventing reaction with OPA (while GSSG is unaffected). Reducing agent is then added to destroy excess quencher and to convert GSSG to GSH. Thus, GSSG can be specifically quantified. The kit provides a unique procedure and buffer formula to eliminate protein thiol interference and to stabilize GSH and GSSG in solution. The assay is easy to perform and detects 2-400 ng/µL of GSH, GSSG or total glutathione.
Comment

Category: Glutathione Assay

Protocol 1. Standard Curve: Add 10 µL of the 20 µg/µL standard GSH stock to 990 µL of Assay Buffer to generate 0.2 µg/µL working standard solution. Add 0, 2, 4, 6, 8, 10 µL to a 96-well plate to generate 0, 0.4, 0.8, 1.2, 1.6, 2.0 µg/well GSH. Bring the volume to 90 µL with Assay Buffer. Note: If the concentration of your assay samples is lower than the above standard range, the standard can be further diluted 10-fold to generate 0, 40, 80, 120, 160, 200 ng/well GSH by following the same procedure.
2. Preparation of Samples for Assays: Add 20 µL of ice cold 3N KOH to 40 µL of PCA preserved samples (as prepared in Section IV) to precipitate PCA and neutralize the samples (pH should be 5-10). Keep on ice for 5 min then spin 2 min at 13,000 G at 4 °C. Transfer 10 µL of the neutralized samples to a 96-well plate. You may choose to add several dilutions (e.g., 1-10 µL) of your samples to ensure the readings are within the standard curve range. A. To Detect GSH: Bring the sample volume to 90 µL with Assay Buffer. B. To Detect Total Glutathione: Bring the sample well to 80 µL with Assay Buffer. Do a background control without sample. Add 10 µL of Reducing Agent Mix to the wells, mix well, incubate at room temperature for 10 min to convert GSSG to GSH. C. To Detect GSSG: Bring the sample well volume to 70 µL with Assay Buffer. Do a background control without sample. Add 10 µL of GSH Quencher, mix well, and incubate at room temperature for 10 min to quench GSH. Then add 10 µL of Reducing Agent Mix to destroy the excess GSH Quencher and convert GSSG to GSH.
3. Assay: Add 10 µL of OPA Probe into the standard and sample wells, mix well, incubate at room temperature for 40 min. Read samples and standards on a fluorescence plate reader equipped with Ex/Em = 340/420 nm. We suggest adjusting the plate reader settings so that the background reading without glutathione is at about 50-150 RFU.
Calculation of Results Calculations: Subtract background reading from sample readings. Plot RFU vs GSH standard. Apply the sample readings to the standard curve to get glutathione amount in each sample. Glutathione Concentration = Ga/Sv Where Ga: Glutathione amount from standard curve.
Restrictions For Research Use only
Storage -20 °C
Expiry Date 12 months
Product cited in: Beauchesne, Desjardins, Butterworth et al.: "Up-regulation of caveolin-1 and blood-brain barrier breakdown are attenuated by N-acetylcysteine in thiamine deficiency." in: Neurochemistry international, Vol. 57, Issue 7, pp. 830-7, 2010 (PubMed).

Guha, Dey, Sen et al.: "Intracellular GSH depletion triggered mitochondrial Bax translocation to accomplish resveratrol-induced apoptosis in the U937 cell line." in: The Journal of pharmacology and experimental therapeutics, Vol. 336, Issue 1, pp. 206-14, 2010 (PubMed).

Bhattacharyya, Tobacman: "Hypoxia reduces arylsulfatase B activity and silencing arylsulfatase B replicates and mediates the effects of hypoxia." in: PLoS ONE, Vol. 7, Issue 3, pp. e33250, 2012 (PubMed).

General Wu, Xu, Velazquez et al.: "Renalase deficiency aggravates ischemic myocardial damage." in: Kidney international, 2010 (PubMed).

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