Glutathione, a tripeptide of glycine, glutamic acid and cysteine, is one of the key antioxidants involved in protecting cells from damages by reactive oxygen species. Glutathione (GSH) reduces disulfide bonds in cytoplasmic proteins to cysteines, in which it is converted to its oxidized form GSSG. GSH/GSSG Assay Kit is designed to accurately measure total, reduced and oxidized glutathione in biological samples using an enzymatic method that utilizes Ellman's Reagent (DTNB) and glutathione reductase (GR). DTNB reacts with reduced glutathione to form a yellow product. The rate of change in the optical density, measured at 412 nm, is directly proportional to glutathione concentration in the sample. This kit can also be used to measure oxidized (GSSG) by using a specific protocol which first scavenges all GSH with 1-methyl-2-vinylpyridinium triflate.||Key Features:|Sensitive and accurate. Linear detection range 0.01-3uM GSH equivalents with a detection limit of 10 nM GSH equivalents.||Applications:|Direct Assays: total, reduced and oxidized glutathione in whole blood, plasma, serum, urine, tissue and cell extracts.|Drug Discovery/Pharmacology: effects of drugs on glutathione metabolism.||Kit Contents: (100 TESTS IN 96-WELL PLATES)|Scavenger: 500ul NADPH: 40ul DTNB: 60ul|2X Assay Buffer: 25ml GR Enzyme: 120ul|Glutathione Standard: 50ul|Storage conditions. The kit is shipped on ice. Store all kit components at -20 °C. Shelf life of six months after receipt.|Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.||Procedures:|Important: equilibrate Scavenger, DTNB and 2X Assay Buffer to room temperature before use. Dilute 2X Assay buffer with an equal volume of dH2O to make 1X Assay Buffer. Briefly mix GR Enzyme before use. Note: b-mercaptoethanol, dithiothreitol and cysteine are known to interfere in this assay. Avoid using these compounds during sample preparation.||Sample Preparation for GSSG Measurement:|Cell lysate can be prepared as follows: wash cells (1-2 x 106) in cold PBS. Lyse cells by homogenization or sonication in 200ul of cold buffer containing 50mM phosphate (pH=7), 1mM EDTA, and 20ul Scavenger. Centrifuge at 10,000g for 5 min at 4°C. Transfer supernatant to a clean tube and proceed to the deproteination procedure. Whole blood samples can be prepared as follows: mix 50ul whole blood with 5ul Scavenger and freeze at -70°C. (Freezing helps lyse the blood cells). After freezing, thaw and mix sample. Incubate at RT for 2-10 min then proceed to the deproteination procedure.||Sample Preparation for Total Glutathione Measurement:|Cell lysate can be prepared as follows: wash cells (1-2 x 106) in cold PBS. Lyse cells by homogenization or sonication in 1ml of cold buffer containing 50mM phosphate (pH=7) and 1mM EDTA. Centrifuge at 10,000g for 15 min at 4°C. Transfer supernatant to a clean tube and proceed to the deproteination procedure. Whole blood samples can be prepared as follows: freeze 50ul whole blood at -70°C. (Freezing helps lyse the blood cells). After freezing, thaw and mix sample. Incubate at RT for 2-10 min then proceed to the deproteination procedure.||Deproteination Procedure:|Prepare a solution of 5wt% Metaphosphoric Acid in water (MPA Reagent). This reagent must be prepared fresh daily. Add 65ul MPA Reagent to 25ul sample, briefly vortex to mix and then centrifuge at 14000 rpm for 5 min. For total glutathione whole blood samples, transfer 5ul of clear supernatant to a clean tube and mix with 620ul 1X Assay Buffer. For all other samples, transfer 6ul of clear supernatant to a clean tube and mix with 244ul 1X Assay Buffer. Transfer 200ul of each neutralized deproteinated sample to separate wells of a 96 well plate.||Glutathione Assay:|1. Standards. First dilute GSH standard to 300uM by mixing 3ul 100mM Standard with 997ul dH2O. Next, prepare the 3uM Premix by mixing 5ul of the 300uM GSH with 495ul 1X Assay Buffer. Dilute standards in 1.5-mL centrifuge tubes as described in the Table.|No Premix+1X Assay Buffer GSH (uM)|1 250ul+0ul 3.0|2 150ul+100ul 1.8|3 75ul+175ul 0.9|4 0ul+250ul 0|Transfer 200ul of each Standard to separate wells in a 96 well plate.|2. Glutathione Detection. Prepare enough working reagent (WR) for 4 standards and all samples. For each reaction combine the following: 105ul 1X Assay Buffer, 1ul GR Enzyme, 0.25ul NADPH and 0.5ul DTNB. Mix WR immediately after adding the DTNB. Add 100ul of WR to each Standard and Sample well. Mix well.|3. Read OD412nm at 0 min and again at 10 min.||Calculation:|Subtract OD0min from OD10min for each Standard and sample. Next subtract the DODBLANK (Std 4) from the DOD values of all Standards and plot the DDOD’s against standard concentrations. Determine the slope using linear regression fitting. The GSSG and GSH concentrations of the Samples are calculated as follows:|[GSHTOTAL] =|DODSAMPLE-DODBLANK|Slope|× n (uM)|[GSH]=[GSHTOTAL]–2 × [GSSG] (uM)|[GSSG]=0.5 ×|DODS(GSSG)-DODBLANK|Slope|× n (uM)|DODSAMPLE, DODBLANK and DODS(GSSG) are the change in optical density values of the sample, water (Std 4) and sample treated with Scavenger, respectively. n is the dilution factor. For all samples treated with Scavenger, n=165. For samples not treated with Scavenger, n=450 for whole blood, and 150 for all other samples.|Conversions: 1 mg/dL glutathione equals 32.5uM, 0.001% or 10 ppm.||Materials Required, But Not Provided:|Pipetting devices, clear flat-bottom 96-well plates, plate reader capable of reading optical density at 412 nm, centrifuge tubes and table centrifuge.|Metaphosphoric Acid can be purchased separately