Frequently Asked Questions for BIOXYTECH®
GSH-400 (Glutathione) Assay
| Catalog Number: 21011|
|Format page for printing|
|How long does the test take to perform?|
|10 minutes for sample preparation.
10 minutes to add reagents and read.
Total procedure time is <25 minutes. |
|What is the sensitivity of the GSH-400 method?|
|The detection limit of the assay is 5 |
|What samples can be tested for glutathione?|
|Any tissue homogenate, cell culture lysate or erythrocyte lysate can be tested.|
|How are samples prepared prior to testing?|
|Any samples containing protein should be treated with metaphosphoric acid. For example, erythrocyte lysates, tissue homogenates, cell culture lysates are treated with metaphosphoric acid. The mixture is centrifuged and the supernatant is tested in the assay. The MPA working solution can be made by dissolving 5g of 33-37% metaphosphoric acid in 100mL H2O. Tissue samples can be minced & homogenized in cold MPA solution. As GSH concentration will vary with the model system the researcher should strive to keep samples as concentrated as possible.|
|What is the stability of the reaction?|
|The chromophore is stable for at least 1 hour if kept in the dark.|
|What control is needed to perform the test?|
|The diluted GSH samples used to prepare the standard curve can be run as controls.|
|What is the shelf life of the GSH-400 kit?|
|12 months after the date of manufacture.
3 months after the vials in the kit are opened. |
|How is glutathione concentration calculated in the procedure?|
|A standard curve is performed by plotting concentration vs. absorbance. The absorbance of the unknown sample is plotted on the standard curve to find its concentration.|
|What are the competitive advantages of the GSH-400 method? Why should I consider a change?|
|Reference technique - requires HPLC; unstable photosensitive reagents; laborious method which takes 90 minutes to 2 hours to read the first sample and 20 minutes to read each sample thereafter.
Ellman Reagent (DTNB) - Not specific; method measures all of the mercaptans in the sample. |
|What are the potential interferences/ pitfalls of this assay?|
|The major source of interference in this assay are proteins. They are eliminated by adding 6% metaphosphoric acid during the precipitation step.
Excess cysteine, if added to a sample (i.e., tissue culture), increases absorbance at 400 nm. This situation is not observed in cell lysates or tissue homogeniates.
|Can I use the GSH-400 with the GSH-420 to measure the GSH/GSSG ratio?|
|The GSH-400 method measures largely the reduced form but some of the GSSG converts to GSH in the assay making it not suitable for this purpose. OXIS R&D personnel have developed the new GSH/GSSG assay.|
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