Glutathione Assay Kit

Catalog No. ABIN1000264

Product Details

Target: Glutathione

Application: Biochemical Assay (BCA)

Sample Type: Blood, Cell Culture Supernatant, Cell Extracts, Plasma, Serum, Urine

Specificity: 12 μg/dL (0.4 μM)

Characteristics: Sensitive and accurate. Linear detection range 0.4 - 100 µM in 96-well plate.
Simple and convenient. The procedure involves mixing the DTNB Reagent with sample, removing protein precipitates for proteinaceous samples, adding a second Reagent and reading the optical density.
Low interference. Amino acids and common buffers do not interfere.

Components: Reagent A: 30 mL. Reagent B: 30 mL. Calibrator: 10 mL (equivalent to 100 µM glutathione).

Material not included: Pipeting devices, centrifuge tube and table centrifuge. Clear bottom 96-well plates (e.g. Corning Costar) and plate reader. Spectrophotometer and cuvets for measuring OD at 412 nm.

Application Details

Application Notes: Direct Assays: reduced glutathione in whole blood, plasma, serum, urine, tissue and cell extracts.
Drug Discovery/Pharmacology: effects of drugs on glutathione metabolism.

Protocol: Procedure using 96-well plate:
1. Blank and Calibrator. Transfer 100 µL water and 100 µL Calibrator into wells of a clear-bottom 96-well plate. Pipette 200 µL water into the Blank and Calibrator wells.
2. Samples. Whole blood samples should be diluted 20-fold with water prior to the assay (n = 20). Deproteination is required for blood, serum, plasma and other proteinaceous samples. Reagent A contains components for both color reaction and deproteination. Mix 120 µL sample with 120 µL Reagent A in1.5-mL centrifuge tubes. Vortex to mix well. If turbidity occurs, pellet 5 min at 14,000 rpm in a table centrifuge. If the mixture remains clear, no centrifugation is necessary.
3. Transfer 200 µL sample/Reagent A mixture into wells of the 96- well plate. Add 100 µL Reagent B. Tap plate lightly to mix.
4. Incubate 25 min at room temperature. Read OD412nm.

Procedure using Cuvet: Mix 400 µL sample with 400 µL Reagent A, centrifuge sample tubes if precipitation occurs. Transfer 600 µL supernatant and mix with 400 µL Reagent B. Incubate 25 min at room temperature. Measure OD412nm against water. Transfer 400 µL Calibrator and 800 µL Water into a clean cuvet and measure OD412nm against water.

Reagent Preparation:

Important: equilibrate Reagents to room temperature. Shake Reagent A before use.

Sample Preparation:

Whole blood samples should be diluted 20-fold with water prior to the assay (n = 20). Cell lysate can be prepared : collect 2 x 10 6 cells by centrifugation at 1,000g for 10 min at 4°C. Wash cells in cold PBS. Lyse cells by homogenization or sonication in 1-2 mL of cold buffer containing 50 mM MES or phosphate (pH 6-7) and 1 mM EDTA. Centrifuge at 10,000g for 15 min at 4°C. Use supernatant for assay. Note: -mercaptoethanol, dithiothreitol and cysteine are known to interfere in this assay. Avoid using these compounds during sample preparation. Amino acids do not interfere.

Calculation of Results:

Subtract blank OD (water) from the Calibrator and Sample OD values.
Conversions: 1 mg/dL glutathione equals 32.5 µM, 0.001% or 10 ppm.

Restrictions: For Research Use only

Handling

Storage: 4 °C

References

Habib, Eisa, Arafat, Marie: "Pulmonary involvement in early rheumatoid arthritis patients." in: Clinical rheumatology, Vol. 30, Issue 2, pp. 217-21, (2011) (PubMed).

Kaur, Sivakumar, Ling: "Melatonin protects periventricular white matter from damage due to hypoxia." in: Journal of pineal research, Vol. 48, Issue 3, pp. 185-93, (2010) (PubMed).

Ogunrinu, Sontheimer: "Hypoxia increases the dependence of glioma cells on glutathione." in: The Journal of biological chemistry, Vol. 285, Issue 48, pp. 37716-24, (2010) (PubMed).

Gumpricht, Devereaux, Dahl, Soden, Sparagna, Leonard, Traber, Sokol: "Resistance of young rat hepatic mitochondria to bile acid-induced permeability transition: potential role of alpha-tocopherol." in: Pediatric research, Vol. 64, Issue 5, pp. 498-504, (2008) (PubMed).

Du, Villeneuve, Wang, Sun, Chen, Li, Lou, Wong, Zhang: "Oridonin confers protection against arsenic-induced toxicity through activation of the Nrf2-mediated defensive response." in: Environmental health perspectives, Vol. 116, Issue 9, pp. 1154-61, (2008) (PubMed).

Target Details

Target: Glutathione

Abstract: Glutathione Products

Synonyms: GT Kit, ILBP Kit, ILLBP Kit, PIP Kit, I-15P Kit, I-BABP Kit, ILBP3 Kit, Illbp Kit, I-BALB Kit, I-BAP Kit, fatty acid binding protein 6 Kit, fatty acid binding protein 6, ileal (gastrotropin) Kit, FABP6 Kit, Fabp6 Kit

Target Type: Chemical

Background: Quantitative determination of reduced glutathione by colorimetric (412nm) method.
Procedure: 30 min.

Glutathione is a tripeptide of glycine, glutamic acid and cysteine. In the red blood cell, the reduced form of glutathione is vital in maintaining hemoglobin in a reduced state and hence protecting the cells from oxidative damage. Glutathione is involved in detoxification of hydrogen peroxide through glutathione oxidase. Low levels of glutathione are found in deficiencies of key enzymes involved in glutathione metabolism, such as glucose-6-phosphate dehydrogenase, glutathione synthase and glutathione reductase. Simple, direct and automation-ready procedures for measuring reduced glutathione are becoming popular in Research and Drug Discovery. This Glutathione Assay Kit is designed to accurately measure reduced glutathione in biological samples. The improved 5,5'-dithiobis(2-nitrobenzoic acid (DTNB) method combines deproteination and detection (Reagent A) into one reagent. DTNB reacts with reduced glutathione to form a yellow product. The optical density, measured at 412 nm, is directly proportional to glutathione concentration in the sample. The optimized formulation has a long shelf life and is completely free of interference due to sample turbidity.