FAQ: Glutathione (GSSG/GSH) Assay

Q: Can this kit be used with samples from any species?

A: Yes, our Total Glutathione Assay is not species specific and should work with any biological protein sample because it measures enzymatic activity rather than protein structure.

 

Q: Is it possible to test samples that have been stored frozen?

A: Two months at -80ºC generally the longest most antioxidant samples should be stored, but we recommend testing fresh samples wherever possible for the best results.

 

Q: Can serum be used with this assay?

A: Serum samples are compatible with the Total Glutathione Assay but the levels are likely to be below the detection limit of the kit.  Better results might be obtained with our Total Antioxidant Capacity Assay #STA-360.

 

Q: Is there any interference from hemoglobin when using liver samples?

A: Hemoglobin will not interfere with the assay, but tissue perfusion is recommended because blood has a high GSH level that can interfere with the assay results.   The product manual contains guidelines for tissue sample preparation.

 

Q: Are samples in EDTA and sulfosalicylic acid compatible with the assay?

A: 5% sulfosalicylic acid is compatible with the assay, although we obtained slightly better results with 5% MPA.  Samples in sulfosalicylic acid and EDTA should be fine in the assay as long as the sulfosalicylic acid is 5%.  Concentrations greater than 5% will inhibit the assay, so samples should be diluted 1:10 with 1X Assay Buffer for a 0.5% final concentration, similar to MPA

 

Q: What is the role of MPA?

A: The metaphosphoric acid (MPA) is used to remove interfering proteins and enzymes from the sample.  It also improves the stability of GSH.

 

Q: Should the 0.5% MPA in assay buffer used for the standard preparation be prepared directly in assay buffer, or from a 1:10 dilution of the 5% in DI water?

A: The 0.5% MPA solution used for diluting the standards can be prepared either way.

 

Q: Where in the protocol is the ethanol used?

A: Ethanol was previously used in an optional protocol and is no longer required for this kit.

 

Q: Can you explain how to make the graph? 

A: Two graphs will be made.  The first graph is OD vs time and this graph will have 8 lines, one for each standard concentration (Figure 2 in the product manual).  The slope is then calculated for each of these lines, resulting in 8 values, one for each standard concentration.  The slope for the 0 standard is used as background and is subtracted from each of the other 7 slopes to give the net slope values.  The net slope values are then plotted on a new graph as net slope vs. concentration (Figure 3 in the product manual).  The unknown samples will be plotted as OD vs. time, similar to the first graph.  After calculating the net slope for each of the unknowns, the values are compared to the second graph (net slope vs. concentration of the standards) to determine the GSSG concentration for each unknown.