ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green)

Transcription

1 ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) Instructions for use: For quantitative measurement of reduced glutathione (GSH/GSSG) in a variety of biological samples. This product is for research use only and is not intended for diagnostic use. Version 20 Last Updated 09 January 2018

2 Table of Contents INTRODUCTION 1 1. BACKGROUND 1 2. ASSAY SUMMARY 2 GENERAL INFORMATION 3 3. PRECAUTIONS 3 4. STORAGE AND STABILITY 3 5. LIMITATIONS 4 6. MATERIALS SUPPLIED 4 7. MATERIALS REQUIRED, NOT SUPPLIED 5 8. TECHNICAL HINTS 6 ASSAY PREPARATION 7 9. REAGENT PREPARATION STANDARD PREPARATION SAMPLE PREPARATION 10 ASSAY PROCEDURE ASSAY PROCEDURE 14 DATA ANALYSIS CALCULATIONS TYPICAL DATA 19 RESOURCES QUICK ASSAY PROCEDURE TROUBLESHOOTING INTERFERENCES FAQS 24

3 INTRODUCTION INTRODUCTION 1. BACKGROUND GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) (ab138881) is a simple, one-step and sensitive assay to quantify reduced or oxidized glutathione in the sample. This kit uses a proprietary non-fluorescent dye that becomes strongly fluorescent upon reacting directly with glutathione, and its signal can be easily read by a fluorescence microplate reader at Ex/Em = 490/520 nm. This product can detect as little as 10 nm of GSH (reduced) or GSSG (oxidized) glutathione (1 pmol/100 µl assay volume). The assay can be performed in a convenient 96-well or 384-well plate format and readily adapted to automation without a separation step. Glutathione is a tripeptide that contains L-cysteine, L-glutamic acid and glycine. It is the smallest intracellular protein thiol molecule in the cells, which prevents cell damage caused by reactive oxygen species such as free radicals and peroxides. Glutathione exists in reduced (GSH) and oxidized (GSSG) states. Reduced glutathione (GSH) is a major tissue antioxidant that provides reducing equivalents for the glutathione peroxidase (GPx) catalyzed reduction of lipid hydroperoxides to their corresponding alcohols and hydrogen peroxide to water. In the GPx catalyzed reaction, the formation of a disulfide bond between two GSH molecules generates oxidized glutathione (GSSG). The enzyme glutathione reductase (GR) recycles GSSG to GSH with the simultaneous oxidation of β-nicotinamide adenine dinucleotide phosphate (β-nadph2). In healthy cells, more than 90% of the total glutathione pool is in the reduced form (GSH). When cells are exposed to increased levels of oxidative stress, GSSG accumulates and the ratio of GSSG to GSH increases. An increased ratio of GSSG-to-GSH is an indication of oxidative stress. The monitoring of reduced and oxidized GSH in biological samples is essential for evaluating the redox and detoxification status of the cells and tissues against oxidative and free radicals mediated cell injury. NOTE: For measuring GSH Standard only, there is enough reagent provided to perform 200 tests. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric - Green) 1

4 INTRODUCTION 2. ASSAY SUMMARY Standard curve preparation Sample preparation* Add Thiol Green Indicator Reaction Mix and incubate RT for minutes Measure fluorescence (Ex/Em = 490/520 nm) *Sample requires deproteinization ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric - Green) 2

5 GENERAL INFORMATION GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. We understand that, occasionally, experimental protocols might need to be modified to meet unique experimental circumstances. However, we cannot guarantee the performance of the product outside the conditions detailed in this protocol booklet. Reagents should be treated as possible mutagens and should be handle with care and disposed of properly. Please review the Safety Datasheet (SDS) provided with the product for information on the specific components. Observe good laboratory practices. Gloves, lab coat, and protective eyewear should always be worn. Never pipet by mouth. Do not eat, drink or smoke in the laboratory areas. All biological materials should be treated as potentially hazardous and handled as such. They should be disposed of in accordance with established safety procedures. 4. STORAGE AND STABILITY Store kit at -20ºC in the dark immediately upon receipt. Kit has a storage time of 1 year from receipt, providing components have not been reconstituted. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in the Materials Supplied section. Aliquot components in working volumes before storing at the recommended temperature. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 3

6 GENERAL INFORMATION 5. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. 6. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) Storage Condition (After Preparation) DMSO 200 µl -20 C -20 C Thiol Green Indicator 1 vial -20 C -20 C Assay Buffer 25 ml -20 C -20 C GSH Standard 1 vial -20 C -20 C (62 µg; lyophilized)* GSSG Standard 1 vial -20 C -20 C (124 µg; lyophilized) GSSG Probe (lyophilized) 1 vial -20 C -20 C *For measuring GSH Standard only, there is enough reagent provided to perform 200 tests. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 4

7 GENERAL INFORMATION 7. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully perform this assay: Microplate reader capable of measuring fluorescence at Ex/Em = 490/520 nm MilliQ water or other type of double distilled water (ddh 2 O) Pipettes and pipette tips, including multi-channel pipette Assorted glassware for the preparation of reagents and buffer solutions Tubes for the preparation of reagents and buffer solutions 96 well plate with clear flat bottom, preferably black Dounce homogenizer (if using tissue) PBS Mammalian Lysis Buffer 5X (ab179835) to homogenize cell or tissue samples. Alternatively, you can PBS/0.5% NP-40 (Optional) Protease inhibitors we recommend Protease Inhibitor Cocktail II (ab201116) [AEBSF, aprotinin, E-64, EDTA, leupeptin] as a general use cocktail Deproteinizing Sample Preparation Kit TCA (ab204708): for deproteinization step in cell and tissues 10 kd Spin Column (ab93349): for deproteinization step in fluid samples ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 5

8 GENERAL INFORMATION 8. TECHNICAL HINTS This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. Selected components in this kit are supplied in surplus amount to account for additional dilutions, evaporation, or instrumentation settings where higher volumes are required. They should be disposed of in accordance with established safety procedures. Avoid foaming or bubbles when mixing or reconstituting components. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Ensure plates are properly sealed or covered during incubation steps. Ensure all reagents and solutions are at the appropriate temperature before starting the assay. Samples which generate values that are greater than the most concentrated standard should be further diluted in the appropriate sample dilution buffer. Make sure you have the right type of plate for your detection method of choice. Make sure all necessary equipment is switched on and set at the appropriate temperature. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 6

9 ASSAY PREPARATION ASSAY PREPARATION 9. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening 9.1. DMSO: Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C 9.2. Assay Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C Thiol Green Indicator 100X: Reconstitute in 100 µl of DMSO (section 9.1) to generate a 100X Stock Solution. Add DMSO drop by drop, vortexing in between, to prevent formation of precipitate. Aliquot dye so that you have enough volume to perform the desired number of assays. Store at -20 C protected from light. Keep on ice while in use GSH Standard (lyophilized, 62 µg): Reconstitute in 200 µl of Assay Buffer to generate a 1 mm (1 nmol/µl) GSH Standard stock solution. Aliquot GSH standard so that you have enough volume to perform the desired number of assays. Store at -20 C protected from light. Keep on ice while in use. NOTE: For measuring GSH Standard only, there is enough reagent provided to perform 200 tests GSSG Standard (lyophilized, 124 µg): Reconstitute in 200 µl of ddh 2 O to generate a 1 mm (1 nmol/µl) GSSG Standard stock solution. Aliquot GSSG standard so that you have enough volume to perform the desired number of assays. Store at -20 C protected from light. Keep on ice while in use GSSG Probe: Reconstitute in 200 µl of ddh 2 O to generate a 25X probe stock solution. Aliquot 25X probe stock solution so that you have enough volume to perform the desired number of assays. Store at -20 C protected from light. Avoid freeze/thaw cycles. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 7

10 ASSAY PREPARATION 10.STANDARD PREPARATION Always prepare a fresh set of standards for every use. Discard the working standard dilutions after use as they do not store well Dilution of GSH standard: Prepare a 10 µm (10 pmol/µl) GSH standard by diluting 5 µl of GSH 1 mm stock into 495 µl of Assay Buffer Using the 10 µm GSH standard, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes: Standard # Sample to Dilute volume standard in well (µl) Assay Buffer (µl) End Conc GSH in well 1 10 µm µm 2 Std # µm 3 Std # µm 4 Std # µm 5 Std # µm 6 Std # µm 7 Std # µm 8 (blank) None µm Each dilution has enough amount of standard to set up duplicate readings (2 x 50 µl). ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 8

11 ASSAY PREPARATION Dilution of GSSG standard: Prepare a 10 µm (10 pmol/µl) GSSG standard by diluting 5 µl of GSSG 1 mm stock into 495 µl of Assay Buffer Using the 10 µm GSSG standard, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes: Standard # Sample to Dilute volume standard in well (µl) Assay Buffer (µl) End Conc GSSG in well 1 10 µm µm 2 Std # µm 3 Std # µm 4 Std # µm 5 Std # µm 6 Std # µm 7 Std # µm 8 (blank) None µm Each dilution has enough amount of standard to set up duplicate readings (2 x 50 µl). NOTE: The concentration of total GSH standard solutions are TWICE the concentrations of GSSG standard solutions. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 9

12 ASSAY PREPARATION 11.SAMPLE PREPARATION General Sample information: We recommend performing several dilutions of your sample to ensure the readings are within the standard value range. We recommend that you use fresh samples. If you cannot perform the assay at the same time, we suggest that you complete the Sample Preparation step before storing the samples. Alternatively, if that is not possible, we suggest that you snap freeze your samples in liquid nitrogen upon extraction and store them immediately at - 80 C. When you are ready to test your samples, thaw them on ice. Be aware however that this might affect the stability of your samples and the readings can be lower than expected. Add protease inhibitors to preparation buffer immediately prior use Cell (adherent or suspension) samples: Harvest the amount of cells necessary for each assay (initial recommendation = cells; initial dilution recommendation = Try starting with ~10,000 to 20,000 cells with adherent cells, and 50,000 to 100,000 cells with suspension cells Wash cells with cold PBS Resuspend cells in 100 µl of ice cold Mammalian Lysis Buffer (alternative, you can use PBS/0.5% NP-40) Homogenize cells quickly by pipetting up and down a few times Centrifuge sample for 15 minutes at 4 C at top speed using a cold microcentrifuge to remove any insoluble material Collect supernatant and transfer to a clean tube Keep on ice. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 10

13 ASSAY PREPARATION Cells samples may contain enzymes that can interfere with the analysis. Remove enzymes from sample by using Deproteinizing Sample Kit TCA (ab204708). Alternatively, you can perform a TCA/ NaHCO 3 deproteinization step following the protocol described in Section Tissue samples: Harvest the amount of tissue necessary for each assay (initial recommendation = 20 mg; initial dilution recommendation = 1/10 1/100) Wash tissue in cold PBS Resuspend tissue in 400 µl of ice cold Mammalian Lysis Buffer (alternatively, you can use PBS/0.5% NP-40) Homogenize tissue with a Dounce homogenizer with passes Centrifuge sample for 15 minutes at 4 C at top speed using a cold microcentrifuge to remove any insoluble material Collect supernatant and transfer to a clean tube Keep on ice Tissue samples may contain enzymes that can interfere with the analysis Remove enzymes from sample by using Deproteinizing Sample Kit TCA (ab204708). Alternatively, you can perform a TCA/ NaHCO 3 deproteinization step following the protocol described in Section Plasma, Serum, Blood and Urine: Biological fluid samples generally contain high amount of proteins which can interfere with the assay. Remove these enzymes from sample by using a 10kD Spin column (ab93349). Add sample to the spin column, centrifuge at 10,000 x g for 10 minutes at 4 C and collect the filtrate. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 11

14 ASSAY PREPARATION Alternative Deproteinization protocol: For this step you will need additional reagents: Trichloroacetic acid (TCA) Sodium bicarbonate (NaHCO3) Prepare samples as specified in protocol. You should have a clear protein sample after homogenization and centrifugation. Keep your samples on ice. Add 1 volume ice cold 100% (w/v) TCA into 5 volumes of sample and vortex briefly to mix well Vortex and incubate on ice for 5 10 minutes Centrifuge samples at 12,000 x g for 5 minutes at 4 C in a cold centrifuge and transfer supernatant to a fresh tube. Measure volume of supernatant. NOTE: samples can be stored at -80 C for 1 month Neutralize the sample by adding NaHCO 3 to supernatant obtained in previous step and vortex briefly. Add NaHCO 3 drop by drop till ph equals 4 6 (use ph paper to test 1 µl of sample). Any leftover TCA will interfere with the assay. NOTE: please avoid adding neutralization buffer to ph > 7 as GSH is very labile and can easily oxidized at ph > Centrifuge at 13,000 x g for 15 minutes at 4 C and collect supernatant Samples are now deproteinized, neutralized and TCA has been removed. The samples are now ready to use in the assay. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 12

15 ASSAY PREPARATION Sample Recovery The deproteinized samples will be diluted from the original concentration. To calculate the dilution factor of your final sample, simply apply the following formula: % original concentration = Initial sample volume (initial sample volume + vol TCA + vol NaHCO 3 ) X100 NOTE: We suggest using different volumes of sample to ensure readings are within the Standard Curve range. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 13

16 ASSAY PROCEDURE ASSAY PROCEDURE 12.ASSAY PROCEDURE Equilibrate all materials and prepared reagents to correct temperature prior to use. We recommended to assay all standards, controls and samples in duplicate. Prepare all reagents, working standards, and samples as directed in the previous sections Set up Reaction Wells GSH Standard wells = 50 µl GSH standard dilutions. GSSG Standard wells = 50 µl GSSG standard dilutions. NOTE: if only GSH is being measured, skip this step. Sample wells = 50 µl samples (adjust volume to 50 µl/well with PBS/0.5% NP-40 or lysis buffer) See example plate layout for reduced glutathione (GSH only) and total glutathione (GSH + GSSG) detection in the table below. Panel A (GSH) Panel B (GSSG) GSH1 GSH1 TS TS GSSG1 GSSG1 TS TS GSH2 GSH2.. GSSG2 GSSG2 GSH3 GSH3 GSSG3 GSSG3 GSH4 GSH4 GSSG4 GSSG4 GSH5 GSH5 GSSG5 GSSG5 GSH6 GSH6 GSSG6 GSSG6 GSH7 GSH7 GSSG7 GSSG7 GSH8 GSH8 GSSG8 GSSG8 GSH= GSH Standards, GSSG= GSSG Standards, TS=Test Samples ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 14

17 ASSAY PROCEDURE GSH Assay Mixture (GAM) [GSH detection]: Prepare GAM by diluting the 100X Thiol Green Stock solution (from step 9.3) in Assay Buffer at 1:100 dilution and mixing well by vortexing. NOTE: GAM solution is stable at 4 C for at least 4 hours when protected from light. For 1x 96-well plate, for instance, dilute 50 µl of 100X Thiol Green stock in 5 ml of Assay Buffer Total GSH Assay Mixture (TGAM) [GSH + GSSG detection]: Prepare TGAM by diluting the 25X GSSG probe stock solution (from step 9.6) in GAM (from step 12.2) at 1:25 dilution and mixing well by vortexing. NOTE: TGAM is unstable at room temperature, and should be used promptly within 2 hours and avoid exposure to light. For 1/2x 96-well plate, for instance, dilute 100 µl 25X GSSG probe stock in 2.5 ml of GAM solution GSH detection: Add 50 µl of GSH Assay Mixture (GAM) into each GSH standard and sample wells (Panel A) to make the total assay volume of 100 µl/well Total GSH + GSSG (reduced and oxidized): Add 50 µl of Total GSH Assay Mixture (TGAM) into each GSSG standard and sample wells (Panel B) to make the total assay volume of 100 µl/well Incubate at room temperature for minutes protected from light Measure output on a fluorescence microplate reader at Ex/Em= 490/520 nm. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 15

18 ASSAY PROCEDURE PROTOCOL FOR 384-WELL PLATE ASSAY Reaction wells: use 25 µl standards and 25 µl sample. Follow same procedure as for 96-well plate till step GSH detection: add 25 µl sample + 25 µl GAM into each well. Total GSH + GSSG: add 25 µl sample + 25 µl TGAM into each well. Proceed with assay as described in step 12.6 ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 16

19 DATA ANALYSIS DATA ANALYSIS 13.CALCULATIONS Samples producing signals greater than that of the highest standard should be further diluted in appropriate buffer and reanalyzed, then multiply the concentration found by the appropriate dilution factor Average the duplicate reading for each standard and sample Subtract the mean fluorescence value of the blank (Standard #8) from all standard and sample readings. This is the corrected fluorescence (fluorescence background increases with time, thus it is important to subtract the fluorescence intensity value of the blank wells for each data point) Plot the corrected values for each standard as a function of the final concentration of GSH and/or Total GSH + GSSG Draw the best smooth curve through these points to construct the standard curve. Most plate reader software or Excel can plot these values and curve fit. Calculate the trendline equation based on your standard curve data (use the equation that provides the most accurate fit) Concentration of reduced glutathione GSH (µm) in the test samples is calculated as: GSH = ( A B ) D Where: A = Amount of GSH in sample well calculated from the GSH standard curve (µmol). B = Sample volume added into the reaction well (µl). D = Sample dilution factor. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 17

20 DATA ANALYSIS Concentration of total glutathione (GSH +GSSG) (µm) in the test samples is calculated as: Total glutathione = ( A B ) D Where: A = Amount of total glutathione (GSH + GSSG) in the sample well calculated from the GSSG standard curve (µmol). B = Sample volume added into the reaction well (µl). D = Sample dilution factor Concentration of oxidized glutathione (GSSG) (µm) in the test samples is calculated as: GSSG = (Total Glutathione GSH)/2 GSH = calculated from step GSH/GSSG Ratio Determination: Ratio = [GSH] / [GSSG] [GSH] = concentration as calculated from step 13.5 [GSSG] = concentration as calculated from step 13.7 ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 18

21 DATA ANALYSIS 14. TYPICAL DATA TYPICAL STANDARD CURVE Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed Figure 1.Typical reduced GSH standard calibration curve after 30 minutes incubation. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 19

22 DATA ANALYSIS Figure 2.Typical total GSH standard calibration curve after 30 minutes incubation. Figure 3. Reduced GSH and total GSH levels in cell lysates. Cells lysed to the concentration of 1x10 7 cells/ml and tested diluted 6-54 fold. *Neuro2a cells were serum starved for 24 hours. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 20

23 RESOURCES RESOURCES 15. QUICK ASSAY PROCEDURE NOTE: This procedure is provided as a quick reference for experienced users. Follow the detailed procedure when performing the assay for the first time. Prepare standard, indicator dye and probe (aliquot if necessary); get equipment ready. Prepare GSH standard dilution [ µm] and (if appropriate) + GSSG standard dilution [ µm]. Prepare samples (including deproteinization step) in optimal dilutions to fit standard curve readings. Set up plate in duplicate for GSH standard (50 µl), GSSG standard (50 µl) and samples (50 µl). Prepare GSH Assay Mixture (GAM) by diluting Thiol Green Indicator in Assay Buffer at 1/100 dilution. Prepare Total GSH Assay Mixture (TGAM) by diluting GSSG probe in GAM solution at 1/25 dilution. Add 50 µl of GAM solution to GSH standard and sample wells. Add 50 µl of TGAM solution to GSSG standard and sample wells. Incubate plate at RT minutes protected from light. Measure fluorescence at Ex/Em= 490/520 nm in a fluorescence microplate reader. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 21

24 RESOURCES 16.TROUBLESHOOTING Problem Cause Solution Assay not working Sample with erratic readings Lower/ Higher readings in samples and Standards Use of ice-cold buffer Plate read at incorrect wavelength Use of a different 96- well plate Samples not deproteinized (if indicated on protocol) Cells/tissue samples not homogenized completely Samples used after multiple free/ thaw cycles Use of old or inappropriately stored samples Presence of interfering substance in the sample Improperly thawed components Allowing reagents to sit for extended times on ice Incorrect incubation times or temperatures Buffers must be at room temperature Check the wavelength and filter settings of instrument Colorimeters: Clear plates Fluorometric: black wells/clear bottom plate Use provided protocol for deproteinization Use Dounce homogenizer (increase number of strokes); observe for lysis under microscope Aliquot and freeze samples if needed to use multiple times Use fresh samples or store at -80 C (after snap freeze in liquid nitrogen) till use Check protocol for interfering substances; deproteinize samples Thaw all components completely and mix gently before use Always thaw and prepare fresh reaction mix before use Verify correct incubation times and temperatures in protocol ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 22

25 RESOURCES Problem Cause Solution Standard readings do not follow a linear pattern Unanticipated results Pipetting errors in standard or reaction mix Air bubbles formed in well Standard stock is at incorrect concentration Measured at incorrect wavelength Samples contain interfering substances Sample readings above/ below the linear range Avoid pipetting small volumes and prepare a master mix whenever possible Pipette gently against the wall of the tubes Always refer to dilutions on protocol Check equipment and filter setting Troubleshoot if it interferes with the kit Concentrate/ Dilute sample so as to be in the linear range ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 23

26 RESOURCES 17. INTERFERENCES These chemicals or biological materials will cause interference in this assay causing compromised results or complete failure: DTT and/or 2-mercaptoethanol: these reducing agents can interfere with the Thiol Green Detection dye and generate fluorescence background. Triton X-100: this product can cause autofluorescence. If using Triton X-100 in the sample preparation buffer, we recommend performing a background control well with preparation buffer without Triton X. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 24

27 RESOURCES 18. FAQs What is the difference between this product and GSH/GSSG Ratio Detection Assay Kit II (Fluorometric Green) (ab205811)? The working principle and detection of both products is the same. The only difference is that the detection probe included in ab205811, Thiol Green Indicator WS is water soluble, whereas the indicator in this product is soluble in DMSO only. Which instrument have you used to test this product? The examples shown in this protocol have been measured using a Gemini fluorescence microplate reader (Molecular Devices). Why is the concentration of GSH twice the concentration of GSSG? The oxidized glutathione (GSSG) will be reduced and formed 2 molecules of reduced glutathione (GSH). 1 mol GSSG = 2 moles GSH The signal I get for GSH is higher than the total glutathione. How is that possible? GSH (reduced glutathione) is very easy to oxidize. If the GSH standard signal is lower than the GSSG signal, or the concentration of GSH in the sample is higher than total glutathione, then GSH has been oxidized. ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 25

28 RESOURCES 19. Notes ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 26

29 RESOURCES ab GSH/GSSG Ratio Detection Assay Kit (Fluorometric Green) 27

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