We have used this kit ony with a UV/Vis spectrophotometer with 1 cm pathlength cuvettes. We have not attempted to modify it for use with a plate reader.
GRSA
Glutathione Reductase Assay Kit
Sufficient for 100 colorimetric tests
Synonym(s):
Glutathione Reductase Activity Assay Kit
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About This Item
UNSPSC Code:
12161503
NACRES:
NA.28
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General description
Glutathione reductase (GR) is a ubiquitous enzyme that catalyzes the reduction of oxidized glutathione (GSSG) to glutathione (GSH). Glutathione reductase is essential for the glutathione redox cycle that maintains adequate levels of reduced cellular GSH, which serves as an antioxidant reacting with free radicals and organic peroxides. Glutathione is also a substrate for the glutathione peroxidases and glutathione S-transferases in the detoxification of organic peroxides and the metabolism of xenobiotics.
Application
Biochem/physiol Actions
Glutathione reductase (EC 1.6.4.2) (GR) is a ubiquitous enzyme that catalyzes the reduction of oxidized glutathione (GSSG) to glutathione (GSH). Glutathione reductase is essential for the glutathione redox cycle that maintains adequate levels of reduced cellular GSH, which serves as an antioxidant reacting with free radicals and organic peroxides. Glutathione is also a substrate for the glutathione peroxidases and glutathione S-transferases in the detoxification of organic peroxides and the metabolism of xenobiotics. This kit contains reagents for the spectrophotometric determination of glutathione reductase activity either by following the decrease in absorbance caused by the oxidation of NADPH at 340 nm (UV assay) or the increase in absorption caused by the reduction of dithiobis(2-nitrobenzoic acid) (DTNB) at 412 nm (colorimetric assay). This kit provides reagents for a spectrophotometric assay for measuring the activity of glutathione reductase either by following the decrease in A340 caused by the oxidation of NADPH or the increase in A412 caused by the reduction of dithiobis(2-nitrobenzoic acid).
This kit provides reagents for a spectrophotometric assay for measuring the activity of glutathione reductase either by following the decrease in A340 caused by the oxidation of NADPH or the increase in A412 caused by the reduction of dithiobis(2-nitrobenzoic acid).
Suitability
Suitable for the measurement of glutathione reductase activity in biological samples
Principle
This kit contains reagents for the spectrophotometric determination of glutathione reductase activity either by following the decrease in absorbance caused by the oxidation of NADPH at 340 nm (UV assay) or the increase in absorption caused by the reduction of dithiobis(2-nitrobenzoic acid) (DTNB) at 412 nm (colorimetric assay). This kit provides reagents for a spectrophotometric assay for measuring the activity of glutathione reductase either by following the decrease in A340 caused by the oxidation of NADPH or the increase in A412 caused by the reduction of dithiobis(2-nitrobenzoic acid).
Analysis Note
UV assay: One unit will cause the oxidation of 1.0 μmole of NADPH at 25 °C at pH 7.5.
Colorimetric assay: One unit will cause the reduction of 1.0 μmole of DTNB to TNB at 25 °C at pH 7.5.
Colorimetric assay: One unit will cause the reduction of 1.0 μmole of DTNB to TNB at 25 °C at pH 7.5.
Storage Class Code
10 - Combustible liquids
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
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Do you know if Product GRSA, Glutathione Reductase Assay Kit, can be used with a 96 well plate reader?
1 answer-
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Is Product GRSA, Glutathione Reductase Assay Kit, compatible with your CelLytic cell lysis reagents?
1 answer-
We have not tried using this kit with our CelLytic reagents. We believe it should be suitable for use with C2978 for mammalian cell lysis or C4882 for yeast cell lysis.
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In your product bulletin you say you have tested Product GRSA, Glutathione Reductase Assay Kit, on rabbit reticulocyte lysate and Saccharomyces extract. Have you tested it on other species?
1 answer-
At this time, we have used the kit to test only these species. We have received inquiries concerning its use for rat and higher plant tissue, but we have no data at this time.
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In your brochure for Nitric Oxide and Cell Stress you present absorbance data from yeast lysis. How were the yeast prepared?
1 answer-
Here is the procedure as recommended by our Production group (March 2008): Grow S. cerevisiae (we used GP460 private strain) on YEPD medium; harvest cells by centrifugation. Wash the cells [7g] with 20 ml of 20 mM HEPES buffer, pH 7.0. Discard supernatant. Add 1 ml HEPES buffer to make a thick slurry and pour dropwise into liquid nitrogen. Take approx. 2 g of frozen pellet and homogenize with UltraTurrax T-25 while still in liquid nitrogen to make a fine powder. After all the liquid nitrogen has evaporated, take approximately 2 mL of HEPES buffer and add to the 2 g of finely powdered cells. Centrifuge for 5 min in an Eppendorf (benchtop) centrifuge at maximum speed. The supernatant liquid is the source of the enzyme. The total amount of protein received from this procedure was 21 mg (8.8 mg/mL in 2.4 mL total volume). We determined an activity of approximately 0.3 units/mL, using both the UV and DTNB assays.
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What is the Department of Transportation shipping information for this product?
1 answer-
Transportation information can be found in Section 14 of the product's (M)SDS.To access the shipping information for this material, use the link on the product detail page for the product.
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How does the storage temperature relate to shipping conditions?
1 answer-
The storage conditions that a Sigma-Aldrich catalog and label recommend for products are deliberately conservative. For many products, long-term storage at low temperatures will increase the time during which they are expected to remain in specification and therefore are labeled accordingly. Where short-term storage, shipping time frame, or exposure to conditions other than those recommended for long-term storage will not affect product quality, Sigma-Aldrich will ship at ambient temperature. The products sensitive to short-term exposure to conditions other than their recommended long-term storage are shipped on wet or dry ice. Ambient temperature shipping helps to control shipping costs for our customers. At any time, our customers can request wet- or dry-ice shipment, but the special handling is at customer expense if our product history indicates that the product is stable for regular shipment.
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Is Product GRSA, Glutathione Reductase Assay Kit, suitable for use for GR determination in whole blood?
1 answer-
We do not recommend the kit for use with whole blood due to interference from hemoglobin.
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