Glutathione Peroxidase Assay Kit, BioAssay(TM)

Glutathione Peroxidase (GPX, EC 1.11.1.9) represents an enzyme family with peroxidase activity whose main biological role is to protect the organism from oxidative damage. It helps prevent lipid peroxidation of cellular membranes by removing free peroxide in the cell. GPX catalyzes the following reaction with glutathione reductase (GR), GPX 2 GSH+H2O2 GS-SG+2 H2O, GR GS-SG+NADPH 2 GSH+NADP+ Simple, direct and high-throughput assays for GPX activity find wide Applications:. The improved assay directly measures NADPH consumption in the enzyme coupled reactions. The measured decrease in optical density at 340nm is directly proportional to the enzyme activity in the sample.\n\nKey Features:\nSensitive and accurate. Use 10ul sample.\nLinear detection range 12 to 300 U/L GPX activity.\n\nApplications:\nDirect Assays: GPX activity in biological samples.\nDrug Discovery/Pharmacology: effects of drugs on GPX activity.\n\nKit Contents: (100 Tests In 96-Well Plates)\nAssay Buffer: 25ml GR Enzyme: 1ml Glutathione: 240ul NADPH: 40ul H2O2 Solution: 100ul C a librator: 100ul Positive Control: 9ul Glutathione Peroxidase (GPX)\nStorage conditions. The kit is shipped on ice. store all components at -20 degrees C. Shelf life of three 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.\n\nSample Preparation:\nAll samples should be clear and free of any turbidity or particles. Liquid samples (e.g. non-hemolyzed serum, plasma) can be assayed directly. Homogenize tissue (10 mg) and cells (106) in 200ul cold 1 x PBS and then centrifuge 10 min at 14,000 rpm to pellet any debris. Use the clear supernatant for the assay. If not assayed immediately, freeze supernatant at -80 degrees C (stable for 1 month).\n\nAssay Procedure:\n1. Reagent Preparation. Equilibrate all components to room temperature. Briefly centrifuge all tubes before opening. Add 360ul dH2O to the NADPH tube (final 35mM). Add 500ul Assay Buffer to the "Positive Control" tube. Vortex tubes to mix. Keep these reconstituted reagent tubes on ice. Unused reagents are stable for three weeks when stored frozen at -20 degrees C.\n2. Standards and Samples. Mix 12ul of the Calibrator with 188ul dH2O (equivalent to 6mM NADPH). Dilute this calibrator stock as shown in the Table below. \nNo 6mM Calibrator+H2O Vol (ul)\n[Equiv. NADPH]\n(mM)\n1 100ul+0ul 100 6.0\n2 60ul+40ul 100 3.6\n3 30ul+70ul 100 1.8\n4 0ul+100ul 100 0\nTransfer 10ul standards into wells of a clear flat-bottom 96-well plate. Add 190ul Assay Buffer to all standard wells. No 6mM Calibrator+H2O Vol (ul) [Equiv. NADPH] (mM) 1 100ul+0ul 100 6.0 2 60ul+40ul 100 3.6 3 30ul+70ul 100 1.8 4 0ul+100ul 100 0 Transfer 10ul sample and 10ul reconstituted GPX Positive Control into separate wells of the 96-well plate. In addition, for each assay run, include a background control that only contains 10ul Assay Buffer. Note: (1). For unknown samples, perform several dilutions to ensure that GPX activity is within the linear range of 12 to 300 U/L. (2) The provided GPX serves as a positive control to ensure assay is working and should not be used to calculate the Sample GPX activity.\n3. Assay. Prepare enough Working Reagent for Sample and Control wells by mixing, for each well, 85ul Assay Buffer, 2ul Glutathione, 2ul 35mM NADPH and 8ul GR enzyme. Add 90ul Working Reagent quickly to the Sample/Control wells. Tap plate to mix. Dilute 8ul 3% H2O2 with 1992ul dH2O (final 3.5mM). Prepare enough 0.35mM H2O2 Reagent by mixing, for each Sample/Control well, 12ul 3.5mM with 108ul dH2O. Use this Reagent within one hour. With a multi-channel pipettor, add 100ul 0.35mM H2O2 Reagent to all Sample and Control wells. Tap plate quickly to mix well contents thoroughly. Immediately read OD340nm (time zero, OD0) and again at 4 min (OD4).\n\nCalculation:\nUse OD values at 4 min for NADPH standards. Subtract blank value (#4) from the standard values. Plot the DOD against standard concentrations and determine the slope of the standard curve. Calculate the DODs=(OD0

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SPECIFICATIONS

Catalog Number

G8127-03

Size

1Kit

References

1. Paglia, D.E. and Valentine, W.N. (1967). Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med. 70: 158-169.\n2. Jacobson, B. et al. (1988). Adaptation of glutathione peroxidase assay to the Technicon RA-1000. Clin Chem. 34: 2164-2165.\n3. Pascual, P. et al. (1992). Direct assay of glutathione peroxidase activity using high-performance capillary electrophoresis. J Chromatogr. 581: 49-56.