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Glutathione (GSH/GSSG/Total) Fluorometric Assay Kit

Details for Product No. ABIN411673, Supplier: Log in to see
Antigen
Reactivity
Amino Acid
21
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1
1
1
1
1
1
1
1
1
1
1
Application
Biochemical Assay (BCA)
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Sample Type Plasma, Serum, Cell Lysate, Tissue Samples, 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 Acid, 6N)
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).
Research Area Metabolism, Amino Acids, Enzymes
Application Notes The kit provides unique formula and buffers and procedures for detecting the GSH, GSSG, and total glutathione individually
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. 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
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