Glutathione Assay Kit (GSH, GSSG and Total)
(Catalog #K264-100;
100 assays; Store kit at -20oC)

I. Introduction:

Glutathione is the major intracellular low-molecular-weight thiol that plays a critical role in cellular defense against oxidative stress in tissues and cells. Commercially available glutathione detection kits, such as the DTNB-enzyme cycling glutathione assay kit or the Monochlorobimane based assay kit hardly distinguish between reduced glutathione (GSH; FW: 307) and oxidized glutathione (GSSG; FW: 612). BioVision’s Glutathione Detection Kit provides a unique, convenient tool for detecting GSH, GSSG, and total glutathione individually. In the assay, OPA, reacts with GSH (not GSSG), generating fluorescence, so GSH can be specifically quantified. Adding a reducing agent converts GSSG to GSH, so (GSH + GSSG) can be determined. To measure GSSG specifically, a GSH Quencher is added to remove GSH, preventing reaction with OPA (while GSSG is unaffected). Reducing agent is then added to destroy excess quencher and to convert GSSG to GSH. Thus, GSSG can be specifically quantified. The kit provides a unique procedure and buffer formula to eliminate protein thiol interference and to stabilize GSH and GSSG in solution. The assay is easy to perform and detects 2-400 ng/l of GSH, GSSG or total glutathione.

II. Kit Contents:

Glutathione Assay Buffer
PCA (Perchloric Acid, 6 N)
KOH (6 N)
OPA Probe (o-phthalaldehyde )
Reducing Agent Mix
GSH Quencher
GSH Standard (FW: 307)

III. Reagent Reconstitution and Storage:

OPA Probe, Reducing Agent Mix, GSH Quencher: Add 0.85 ml H2O to the OPA probe, mix. Dissolve both the Reducing Agent and the GSH Quencher in 1.05 ml of dH2O separately. Store at –20oC. Use within two months. GSH Standard: Accurately dissolve in 45 l dH2O and add 5 l PCA to stabilize the standard GSH stock solution (20 ug/ul). Store at –20oC. Use within two months.

IV. Sample Collection and Storage*:

Tissue and Cell Samples:
GSH is labile and cell preparations will oxidize it rapidly. Keep all samples and reagents ice cold and work as rapidly as possible. Prepare centrifuge tubes with 20 μl PCA on ice to receive samples. Homogenize 2-4 x106 cells or 40 mg tissue on ice with 100 l of ice cold Glutathione Assay Buffer. Take 60 l of each homogenate to a prechilled tube containing PCA and vortex several seconds to achieve a uniform emulsion. Keep on ice for 5 min. Spin 2 min at 13,000 G at 4oC, collect supernatant (containing glutathione) and discard the protein pellet. The sample can then be stored at –80oC, stable for a month.
Serum or Other Liquid Samples:
Freeze samples immediately upon acquisition and keep frozen until ready for processing. Take 60 l thawed sample to centrifuge tube containing 20 l ice cold PCA, vortex and keep on ice for 5 min. Spin for 2 min at 13,000 G at 4oC. Collect the supernatant. The sample can then be stored at –80oC, stable for a month.

V. Assay Protocol:

1. Standard Curve: Add 10 ul of the 20 ug/ul standard GSH stock to 990 ul of Assay Buffer to generate a 0.2 ug/ul working standard solution. Add 0, 2, 4, 6, 8, 10 ul to a 96-well plate to generate 0, 0.4, 0.8, 1.2, 1.6, 2.0 ug/well GSH. Bring the volume to 90 ul with Assay Buffer.
Note: If the concentration of your assay samples is lower than the above standard range, the standard can be further diluted 10-fold to generate 0, 40, 80, 120, 160, 200 ng/well GSH by following the same procedure.
2. Preparation of Samples for Assays: Add 20 ul of ice cold 3N KOH to 40 ul of PCA preserved samples (as prepared in Section IV) to precipitate PCA and neutralize the samples (pH should be 5-10). Keep on ice for 5 min then spin 2 min at 13,000 G at 4oC. Transfer 10 ul of the neutralized samples to a 96-well plate. You may choose to add several dilutions (e.g., 1-10 ul) of your samples to ensure the readings are within the standard curve range.
A. To Detect GSH: Bring the sample volume to 90 ul with Assay Buffer.
B. To Detect Total Glutathione: Bring the sample well to 80 ul with Assay Buffer. Do a background control without sample. Add 10 ul of Reducing Agent Mix to the wells, mix well, incubate at room temperature for 10 min to convert GSSG to GSH.
C. To Detect GSSG: Bring the sample well volume to 70 ul with Assay Buffer. Do a background control without sample. Add 10 ul of GSH Quencher, mix well, and incubate at room temperature for 10 min to quench GSH. Then add 10 ul of Reducing Agent Mix to destroy the excess GSH Quencher and convert GSSG to GSH.
3. Assay: Add 10 ul of OPA Probe into the standard and sample wells, mix well, incubate at room temperature for 40 min. Read samples and standards on a fluorescence plate reader equipped with Ex/Em = 340/420 nm. We suggest adjusting the plate reader settings so that the background reading without glutathione is at about 50-150 RFU.
4. Calculations: Subtract background reading from sample readings. Plot RFU vs GSH standard. Apply the sample readings to the standard curve to get glutathione amount in each sample.
Glutathione Concentration = Ga/Sv
Where Ga: Glutathione amount from standard curve.
Sv: Sample volume added to the sample wells (Note: The original samples have been diluted 2-fold by performing the procedures above).