OxiSelect™ Glutathione Reductase Assay Kit
Contents
1. Product Manual OxiSelect Glutathione Reductase Assay Kit Catalog Number STA 812 100 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Glutathione reductase is a homodimeric enzyme that is a member of the flavoprotein disulfide oxidoreductases It has an indirect impact in the prevention of oxidative damage in cells by helping to maintain intracellular reduced glutathione GSH Thus measuring the activity of the enzyme is an indicator of oxidative stress It is a ubiquitous enzyme that catalyzes the NADPH dependent reduction reaction of oxidized glutathione GSSG to reduced glutathione GSH Oxidized glutathione is reduced through a multi step reaction in which glutathione reductase is reduced by NADPH which in turn reacts with aGSSG molecule This creates a disulfide interchange reaction that creates two GSH molecules and restores glutathione reductase to its oxidized form Regenerated GSH is available to detoxify hydrogen peroxide Maintenance of GSH is vital in oxidation reduction processes as well as detoxification of hydrogen peroxide and organic peroxides brought on by inflammation in cells Cell Biolabs OxiSelect Glutathione Reductase Assay Kit is a quantitative assay for measuring glutathione reductase activity within plasma erythrocytes tissues and cell lysates Glutathione reductase activity is defined as 1 unit of e2. Store the remaining kit components at 4 C Preparation of Reagents e 1X Assay Buffer Prepare 1X Assay Buffer by adding 200 mL of deionized water to 50 mL of the 5X Assay Buffer Mix thoroughly until homogeneous Use this buffer for preparing kit reagents Store at 4 C when not in use e Chromogen Prepare the Chromogen just before use and prepare only enough for immediate applications Dilute the Chromogen stock 1 15 with 1X Assay Buffer eg Add 200 uL of Chromogen stock to 2 8 mL of 1X Assay Buffer Vortex thoroughly e 1X NADPH Prepare 1X NADPH by diluting the stock solution 1 50 with 1X Assay Buffer Vortex the stock tube thoroughly prior to preparing Prepare only enough for immediate applications eg Add 25 uL of NADPH stock to 1 225 mL Assay Buffer Preparation of Samples These preparation protocols are intended as a guide for preparing known samples The user may need to adjust the sample treatment accordingly All samples should be assayed immediately or stored for up to 1 2 months at 80 C A trial assay with a representative test sample should be assayed to determine the samples compatibility with the dynamic range of the standard The assay can be used on cell culture supernatants plasma erythrocytes tissues and cell lysates High levels of interfering substances may cause variations in results Run proper controls as necessary Always run a standard curve with samples Notes e Thiol compounds such as cysteine dithi
3. 1 2 months Dilute with 1X Assay Buffer as necessary before testing Preparation of Standard Curve 1 To prepare glutathione reductase standards first perform a 1 500 dilution of the stock Glutathione Reductase in 1X Assay Buffer Use only enough for immediate applications eg Add 5 uL of Glutathione Reductase to 2495 uL 1X Assay Buffer This solution has a concentration of 1 Unit mL or 1000 mU mL Use microfuge tubes to prepare a series of standards according to Table 1 below Prepare standards fresh for each assay performed Vortex tubes thoroughly when preparing as the glutathione reductase solution is a suspension and therefore will settle upon standing Do not store or reuse standard preparations 1000 mU mL Glutathione Reductase Glutathione Standard Standard 1X Assay Buffer Reductase Tubes uL uL mU mL 1 80 920 80 2 500 of Tube 1 500 40 3 500 of Tube 2 500 20 4 500 of Tube 3 500 10 5 500 of Tube 4 500 5 6 500 of Tube 5 500 2 5 7 500 of Tube 6 500 1 25 8 0 500 0 Table 1 Preparation of Glutathione Reductase Standards Assay Protocol 1 Qua qe tuy opa Prepare and mix all reagents thoroughly before use Prepare the glutathione reductase standards simultaneously with the samples so they may be assayed together Each sample including unknown and standard should be assayed in duplicate or triplicate In a 96 well plate add 25 uL of the 1X NADPH solut
4. RANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2014 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing
5. al Glutathione Assay Kit OxiSelect TBARS Assay Kit MDA Quantitation OxiSelect Superoxide Dismutase Activity Assay OxiSelect Catalase Activity Assay Kit OxiSelect Intracellular ROS Assay Kit Green Fluorescence OxiSelect In Vitro ROS RNS Assay Kit Green Fluorescence OxiSelect ORAC Activity Assay OxiSelect MDA Competitive ELISA Kit OxiSelect HNE Adduct Competitive ELISA Kit Kit Components Box 1 shi 1 2 3 4 Assay Buffer 5X Part No 281204 Two 25 mL bottles Box 2 shi ed at room temperature Glutathione Reductase Standard Part No 281201 One 20 uL amber tube Glutathione Disulfide GSSG Part No 281202 One 5 mL bottle of a 10 mM solution Chromogen Part No 281203 One 0 5 mL amber tube ed on blue ice packs 1 NADPH 50X Part No 231203 One 50 uL amber tube Materials Not Supplied 1 om NAA KR wD 96 well microtiter plate Distilled or deionized water 1X PBS 10 uL to 1000 uL adjustable single channel micropipettes with disposable tips 50 uL to 300 uL adjustable multichannel micropipette with disposable tips Conical tubes and bottles for sample and buffer preparation Centrifuge and or microfuge Sonicator or tissue homogenizer Multichannel micropipette reservoirs 10 Ethanol 11 Microplate reader capable of reading 405 nm Storage Upon receipt store the NADPH at 80 C Prepare single use aliquots and avoid multiple freeze thaw cycles
6. ion to each well to be tested Add 100 uL of the prepared glutathione reductase standards or samples to each well to be tested Add 50 uL of the 1X Chromogen and mix briefly Ensure that the plate reader is prepared for a kinetic assay and is set to read at 405 nm Add 25 uL of the Glutathione Disulfide GSSG solution and mix briefly Immediately begin recording the absorbance at 405 nm at 1 minute intervals for 10 minutes The reaction may be run longer if necessary If using all the wells within the plate at one time then it may be necessary to record the absorbance at 2 minute intervals Calculate the concentration of standards and samples See Calculation of Results below Calculation of Results l 2 First determine the average of the replicate absorbance readings for each glutathione reductase standard sample and negative control for every time point taken Graph the average of each standard sample and background absorbance at 405nm against incubation time Determine the slope for each value from the linear portion of each curve See Figure 2 under Example of Results below Next subtract the background slope from the slope of the standards and samples Plot the net slopes of the glutathione reductase standards against the microunits mL concentration of glutathione reductase See Figure 3 under Example of Results below Compare the net slopes of the samples with the standard curve from Figure 3 and determine the microu
7. nits mL concentration of glutathione reductase for each sample Note Remember that the standard and samples are diluted 1 2 dilution within the final assay reaction protocol Example of Results The following figures demonstrate typical Glutathione Reductase Assay results at 405 nm One should use the data below for reference only This data should not be used to interpret actual results Glutathione Reductase mU mL OD 405 nm 6 Time Minutes Figure 2 Glutathione Reductase Standard Curve OD 405nm versus incubation time as a function of Glutathione Reductase concentration 0 09 0 08 0 07 0 06 0 05 0 04 0 03 0 02 0 01 0 Net Slope OD 405 nm min 0 5 10 15 20 25 Glutathione Reductase mU mL Figure 3 Glutathione Reductase Standard Curve Net slope versus Glutathione Reductase concentration References 1 2 3 4 Anderson M Glutathione in Free radicals A Practical Approach Oxford University Press New York 1996 Halliwell B Gutteridge J M C Free Radicals in Biology and Medicine Oxford University Press New York 1999 Julius M et al J Clin Epidemiol 1994 47 1021 1026 Mytilineou C et al Parkinsonism Relat Disord 2002 8 385 387 Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WAR
8. nzyme reducing 1 umole oxidized glutathione GSSG per minute at pH 7 6 and 25 C The kit employs a simple enzymatic recycling reaction for glutathione quantification where the reduction of a chromagen is correlated to glutathione reductase enzymatic activity The kit has a detection sensitivity limit of approximately 0 6 mU mL Each kit provides sufficient reagents to perform up to 100 assays including standard curve and unknown samples Assay Principle The OxiSelect Glutathione Reductase Assay Kit is a quantitative assay for measuring the glutathione reductase activity within a sample Glutathione Reductase reduces oxidized glutathione GSSG to reduced glutathione GSH in the presence of NADPH Subsequently the chromogen reacts with the thiol group of GSH to produce a colored compound that absorbs at 405 nm Figure 1 The glutathione reductase content in unknown samples is determined by comparison with the predetermined glutathione reductase standard curve The rate of chromophore production is proportional to the concentration of glutathione reductase activity within the sample The rate can be determined from the absorbance change over time NADPH GSSG OD 405nm Glutathione Reductase NADP 2GSH Chromogen Figure 1 Assay Principle Related Products 1 CoN DANA wWHN STA 310 STA 312 STA 330 STA 340 STA 341 STA 342 STA 347 STA 345 STA 832 10 STA 838 OxiSelect Protein Carbonyl ELISA Kit OxiSelect Tot
9. o lyse the cells Centrifuge at 10 000 12 000 rpm for 10 minutes at 4 C Collect the supernatant Store on ice if assaying immediately or freeze at 80 C for up to 1 2 months Dilute with 1X Assay Buffer before testing Cell Lysates Wash adherent cells at 2 6 x 106 cells with IX PBS Harvest adherent cells with a rubber policeman or gentle trypsinization Centrifuge adherent or suspension cells at 500 1000 rpm for 5 minutes at 4 C Remove supernatant and wash cells in cold PBS Repeat centrifugation and remove solution Immediately resuspend the cell pellet with 200 500 uL ice cold 1X Assay Buffer for a cell number of 1 5 x 10 cells Mix thoroughly Homogenize or sonicate cell suspension and store on ice until use Transfer the suspension to a microfuge tube and centrifuge at 12 000 rpm for 5 minutes at 4 C Collect the supernatant Store on ice if used immediately or freeze at 80 C for up to 1 2 months Dilute with 1X Assay Buffer as necessary before testing Tissue Lysate Perfuse or wash the tissue to remove blood cells and clots with a cold PBS 1 mM EDTA Homogenize the tissue thoroughly with cold isotonic saline solution of 1X PBS with 0 16 mg mL heparin to prevent coagulation Blot the tissue dry and weigh Add ice cold PBS I mM EDTA 1 mL 100 mg tissue and homogenize using a glass pestle Centrifuge the homogenate at 12 000 rpm for 15 minutes at 4 C Collect the supernatant Store on ice if used immediately or freeze at 80 C for up to
10. othreitol DTT or B mercaptoethanol can interfere with the assay by competing with GSH for binding to the chromogen In addition N ethylmaleimide or other thiol alkylating reagents should also be avoided because they will interfere with glutathione reductase and GSH e Make serial dilutions of samples as necessary to obtain a quantifiable change in absorbance readings over time e Samples containing reduced glutathione GSH should be accounted for by running the sample without Glutathione Disulfide GSSG and subtracting these background values from a treated sample A kinetic assay is recommended because it is more precise than an end point assay e Plasma Add blood sample to a blood collection tube with an anticoagulant such as heparin EDTA or sodium citrate Centrifuge at 3 000 rpm for 10 15 minutes at 4 C Remove the upper yellow plasma supernatant layer without disturbing the white buffy coat leukocytes Store on ice if assaying immediately or freeze at 80 C for up to 1 2 months Dilute the plasma with 1X Assay Buffer before testing e Erythrocytes Add blood sample to a blood collection tube with an anticoagulant such as heparin EDTA or sodium citrate Centrifuge the blood at 3 000 rpm for 10 15 minutes at 4 C Remove the white interface buffy coat leukocytes and yellow plasma layers and discard Wash the erythrocytes in cold PBS or saline Determine the packed cell volume and add 4 cell volumes of 4 cold deionized water t
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