Glutathione Colorimetric Detection Kit

Allow the kit reagents to come to room temperature for 30 minutes.
We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine GSH concentrations.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Sample Diluent Prepare the Sample Diluent by diluting one part 5 % SSA 1:5 with four parts Assay Buffer and vortex thoroughly.
The pH of the Sample Diluent must be > 6.
Sample Diluent can be stored at 4 °C for one month. 2-Vinylpyridine treatment To measure Oxidized Glutathione, free GSH must be blocked by alkylation.
To 250 μL of SSA treated samples, standards or Sample Diluent add 5 μL of the ethanolic solution of 2VP (see page 6) and allowed to incubate at room temperature for 1 hour.
The 2VP treated samples and standards should then be diluted in Assay Buffer and Sample Diluent according to the dilutions recommended for each sample type on pages 7 and 8 prior to using in the assay.
The 2VP treated Sample Diluent is used for the zero standard on page 11.
Samples treated with 2VP should be read off a standard curve generated with 2VP treated standards.
Colorimetric Detection reagent Prepare the Colorimetric Detection Reagent by diluting one part Colorimetric Detection Reagent Concentrate 1:10 with nine parts Assay Buffer.
See Colorimetric Detection Reagent Dilution Table for suitable volumes.
Colorimetric Detection reagent Dilution table 1/2 Plate one Plate two Plates four Plates Colorimetric Detection Concentrate 140 μL 260 μL 500 μL 1 mL Assay Buffer 1.26 mL 2.34 mL 4.5 mL 9 mL total Colorimetric reagent Volume 1.4 mL 2.6 mL 5 mL 10 mL reaction Mixture Prepare the Reaction Mixture by diluting one part each NADPH and Glutathione Reductase Concentrates 1:10 into eight parts Assay Buffer.
See Reaction Mix Dilution Table for suitable volumes.
Store any unused Reaction Mixture at 4 °C for no more than 2 days. reaction Mix Dilution table 1/2 Plate one Plate two Plates four Plates nADPh Concentrate 140 μL 260 μL 500 μL 1 mL Glutathione reductase Concentrate 140 μL 260 μL 500 μL 1 mL Assay Buffer 1.12 mL 2.08 mL 4 mL 8 mL total reaction Mix Volume 1.4 mL 2.6 mL 5 mL 10 mL ® www.ArborAssays.com 9 Standard Preparation for the measurement of oxidized Glutathione (GSSG), a 50 μL aliquot of the 250 μM Oxidized Glutathione Standard should be treated with 1 μL of 2VP as outlined on page 9. 2VP-treated Standards are prepared by labeling test tubes as #1 through #6.
Pipet 475 μL of Sample Diluent into tube #1 and 250 μL into tubes #2 to #6.
Carefully add 25 μL of the 2VP-treated Standard to tube #1 and vortex completely.
Take 250 μL of the solution in tube #1 and add it to tube #2 and vortex completely.
Repeat this for tubes #3 through #6.
The concentration of Oxidized Glutathione in tubes 1 through 6 will be 12.5, 6.25, 3.125, 1.56, 0.781 and 0.391 μM.
The concentration of Total GSH in tubes 1 through 6 will be 25, 12.5, 6.25, 3.125, 1.56, and 0.781 μM after addition of the Reaction Mixture. 2VP treated Sample Diluent must be used as a 0 μM standard. to determine total GSh, the Standards are prepared by labeling test tubes as #1 through #6.
Pipet 475 μL of Sample Diluent into tube #1 and 250 μL into tubes #2 to #6.
Carefully add 25 μL of the supplied Standard to tube #1 and vortex completely.
Take 250 μL of the solution in tube #1 and add it to tube #2 and vortex completely.
Repeat this for tubes #3 through #6.
The concentration of Total GSH in tubes 1 through 6 will be 25, 12.5, 6.25, 3.125, 1.56, and 0.781 μM after addition of the Reaction Mixture.
Sample Diluent must be used as a 0 μM standard. use all Standards within 2 hours of preparation.
Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Sample Diluent Volume (μl) 475 250 250 250 250 250 Addition Stock Std 1 Std 2 Std 3 Std 4 Std 5 Volume of Addition (μl) 25 250 250 250 250 250 GSSG Conc (μM) 12.5 6.25 3.125 1.56 0.781 0.391 total GSh Conc (μM) 25 12.5 6.25 3.125 1.56 0.781 ® 10 EXPECT ASSAY ARTISTRY ASSAy ProtoCol - enD Point for oxidized Glutathione (GSSG) use the 2VP treated standards, 2VP treated Sample Diluent and 2VP treated samples diluted with Sample Diluent as described previously. for total Glutathione use the standards and samples diluted with Sample Diluent as described previously. 1.
Use the plate layout sheet on the back page to aid in proper sample and standard identification. 2.
Pipet 50 μL of either 2VP treated or untreated samples or standards into duplicate wells in the plate. 3.
Pipet 50 μL of either 2VP treated or untreated Sample Diluent into duplicate wells as the Zero standard. 4.
Add 25 μL of the Colorimetric Detection Reagent to each well using a repeater pipet. 5.
Add 25 μL of the Reaction Mixture to each of the wells using a repeater pipet. 6.
Gently tap the sides of the plate to ensure adequate mixing of the reagents. 7.
Incubate at room temperature for 20 minutes. 8.
Read the optical density at 405 nm.
These data will be used to determine either Oxidized Glutathione or Total Glutathione concentration.
ASSAy ProtoCol - KinetiC 1.
Carry out steps 1-4 above. 2.
Gently tap the sides of the plate to ensure adequate mixing of the reagents. 3.
Add 25 μL of the Reaction Mixture to each of the wells using a repeater and immediately place plate in reader and read optical density at 405 nm every minute for at least 10 minutes.
These data will be used to determine Total or Oxidized Glutathione concentration kinetically.

All samples must be treated with the SSA solution prepared on page 6. All of the SSA treated centrifuged supernatants must have their SSA concentration brought down to 1 % SSA by dilution with Assay Buffer. Further dilutions of the sample, using Sample Diluent (see page 9 for preparation), may be necessary to allow the GSH concentration to be measurement in the assay. Detailed instructions follow. All samples and standards must be in Sample Diluent before starting the assay To measure Oxidized Glutathione in samples, reduced Glutathione (GSH) in the sample must be blocked by treatment with 2-vinylpyridine, 2VP (see page 6 for preparation). SSA treated samples should be treated with 2VP by addition of 5 μL of 2VP solution for every 250 μL of sample (see page 9). 2VP treated samples must be read off a standard curve made with 2VP-treated standards. use all samples within 2 hours of dilution. Whole Blood, Serum, eDtA or heparin Plasma, or urine Thoroughly mix sample with an equal volume of cold 5 % SSA. Incubate for 10 minutes at 4 °C. Centrifuge at 14,000 rpm for 10 minutes at 4 °C. Collect the supernatant. If the supernatent contains particulates, re-centrifuge the supernatant for 15 minutes and collect the clarified second supernatant. Samples can be stored in aliquots at ≤ -70 °C or analyzed immediately. At this point the SSA concentration will be 2.5 %. The supernatant must be diluted 1:2.5 with Assay Buffer by mixing one part with 1.5 parts of Assay Buffer to bring the SSA concentration to 1 %. The sample will have been diluted 1:5 at this point. All final dilutions are made in Sample Diluent. Treated Whole Blood must be further diluted at least 1:20 for a recommended final dilution of ≥ 1:100. For Treated Plasma and Treated Urine a final dilution of ≥ 1:5 is recommended, but further dilutions in Sample Diluent may be necessary.

Average the duplicate optical density readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean ODs for the zero standard.
The concentrations obtained should be multiplied by the dilution factor to obtain sample values.