Glutathione Colorimetric Assay Kit Kit
- Detection Method
- Detection (D)
- Sample Type
- Cell Lysate, Plasma, Serum, Tissue Samples
- 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.
- ApoGSHTM Glutathione Colorimetric Detection Kit: Convenient & Sensitive Colorimetric Assay for analyzing either Total Glutathione or Reduced form of Glutathione from Tissues, Cells, Plasma & Erythrocytes.
Glutathione Reaction Buffer
Glutathione Substrate (DTNB)
NADPH Generating Mix (lyophilized)
Glutathione Reductase (lyophilized)
Sulfosalicylic Acid (SSA, 1 gram)
GSH Standard (lyophilized, MW 307)
Xanthine/Hypoxanthine Colorimetric/Fluorometric Assay Kit Kit
D, FM Reactivity: Chemical Fluorometric, Colorimetric Adherent Cell Culture, Cell Culture Cells, Cell Culture Supernatant, Cell Samples, Plasma, Serum, Tissue Samples
- Application Notes
- The Colorimetric Glutathione Detection Kit provides a simple in vitro assay for detecting the GSH changes in apoptosis and other pathological processes.
Further details regarding sample type: Cell and tissue lysates, culture media, urine, plasma and serum, as well as many other biological fluids
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.
- For Research Use only
- -20 °C
- Expiry Date
- 12 months
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- 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
- 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.