Glutathione (GSH) Assay Kit, BioAssay™

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. 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 412nm, 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.||Key Features:|Sensitive and accurate. Linear detection range 0.4-100uM 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.||Applications:|Direct Assays: reduced glutathione in whole blood, plasma, serum, urine, tissue and cell extracts.|Drug Discovery/Pharmacology: effects of drugs on glutathione metabolism.||Kit Components: |(250 tests in 96-well plates)|G8123-47A: Reagent A, 1x30ml |G8123-47B: Reagent B, 1x30ml |G8123-47C: Calibrator, 100uM, 1x10ml (equivalent to 100uM glutathione)||Storage and Stability:|Store components at 4°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. ||Protocol:|Important: equilibrate Reagents to room temperature. Shake Reagent A before use.|Samples: whole blood samples should be diluted 20-fold with water prior to the assay (n=20). Cell lysate can be prepared as follows: collect 2 x 106 cells by centrifugation at 1,000g for 10 min at 4°C. Wash cells in cold PBS. Lyse cells by homogenization or sonication in 1-2ml of cold buffer containing 50mM MES or phosphate (pH 6-7) and 1mM EDTA. Centrifuge at 10,000g for 15 min at 4°C. Use supernatant for assay.|Note: b-mercaptoethanol, dithiothreitol and cysteine are known to interfere in this assay. Avoid using these compounds during sample preparation. Amino acids do not interfere.|Procedure using 96-well plate:|1. Blank and Calibrator. Transfer 100ul water and 100ul Calibrator into wells of a clear-bottom 96-well plate. Pipette 200ul 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 120ul sample with 120ul Reagent A in 1.5-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 200ul sample/Reagent A mixture into wells of the 96- well plate. Add 100ul Reagent B. Tap plate lightly to mix.|4. Incubate 25 min at room temperature. Read OD412nm.|Procedure using Cuvet:|Mix 400ul sample with 400ul Reagent A, centrifuge sample tubes if precipitation occurs. Transfer 600ul supernatant and mix with 400ul Reagent B. Incubate 25 min at room temperature. Measure OD412nm against water. Transfer 400ul Calibrator and 800ul Water into a clean cuvet and measure OD412nm against water.||Calculation:|Subtract blank OD (water) from the Calibrator and Sample OD values. The glutathione concentration of Sample is calculated as|=|ODSAMPLE–ODBLANK|ODCALIBRATOR–ODBLANK|X 100 x n (uM)|ODSAMPLE, ODSTD and ODBLANK are optical density values of the sample, Calibrator and sample “Blank” (water or buffer in which the sample was dissolved). n is the dilution factor (20 for blood samples).|Conversions: 1 mg/dL glutathione equals 32.5uM, 0.001% or 10 ppm.||Materials Required, But Not Provided:|Pipeting devices, centrifuge tube and table centrifuge.|Procedure using 96-well plate:|Clear bottom 96-well plates (e.g. Corning Costar) and plate reader.|Procedure using cuvette:|Spectrophotometer and cuvets for measuring OD at 412 nm.||Example:|20ul fresh mouse blood was mixed quickly with 380ul water. Assays in 96-well plate gave blood glutathione concentration of 1124 ± 8uM (n=2)