-
Stock availability
In Stock
-
Kit s description
Fluorometric detection of glutathione reductase activity by the amount of GSH generated from the reduction of GSSG
-
Protein target
Glutathione Reductase
-
Scientific context
Glutathione reductase (GR) plays an indirect but essential role in the prevention of oxidative damage within the cell by helping to maintain appropriate levels of intracellular glutathione (GSH). GSH, in conjuction with the enzyme glutathione peroxidase (GP), is the acting reductant responsible for minimizing harmful hydrogen peroxide cellular levels (1). The regeneration of GSH is catalyzed by GR (2). GR is an ubiquitous 100-120 kDa dimeric flavoprotein that catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione, using ß-nicotinamide dinucleotide phosphate (NADPH) as the hydrogen donor (3). Molecules such as NADPH act as hydride donors in a variety of enzymatic processes. NADPH has been suggested to also act as an indirectly operating antioxidant, given its role in the re-reduction of GSSG to GSH and thus maintaining the antioxidative power of glutathione.
-
Primary research area
Cancer, Oxidative Stress
-
Category
Assay Activity Kits
-
Other name
γ-L-Glutamyl-L-cysteinylglycine Activity Kit (2S)-2-Amino-5-[[(2R)-1-(carboxymethylamino)-1-oxo- 3-sulfanylpropan-2-yl]amino]-5-oxopentanoic acid Activity Kit
-
Brand name
StressXpress®
-
Detection system
Fluorometric Assay
-
Assay format
Direct Enzyme Activity Assay
-
Main intended usage
Fluorometric assay used to determine glutathione reductase activity by the amount of GSH generated from the reduction of GSSG, in samples.
-
Brief protocol
The Glutathione Reductase Activity kit is designed to quantitatively measure glutathione reductase (GR) activity in a variety of samples. A GR standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. The kit utilizes a proprietary non-fluorescent molecule that will covalently bind to the free thiol group on GSH generated in the reduction of oxidized glutathione (GSSG) to yield a highly fluorescent product. After mixing the sample or standard with StressXpress® Detection Reagent and incubating at room temperature, the fluorescent product is read at 510 nm in a fluorescent plate reader with excitation at 390 nm. Background thiol content is read first after 5 minutes, followed by addition of GSSG and NADPH which uses the standard or sample GR to convert the oxidized glutathione, GSSG, into free GSH, which then reacts with the StressXpress® Detection Reagent to yield the signal related to GR activity The activity of GR in the sample is calculated from the generated signal. We have provided a 96 well plate for measurement but this assay is adaptable for higher density plate formats. The end user should ensure that their HTS black plate is suitable for use with these reagents prior to running samples.
-
Kit contents
Black Half Area 96 Well Plate, Glutathione Reductase Standard, StressXpress® Detection Reagent, Dry DMSO, Assay Buffer Concentrate, NADPH, NADPH Diluent, Oxidized Glutathione
-
Assay precision
Intra Assay Precision: Five native samples diluted in Assay Buffer and run in replicates of 16 in an assay. The mean and precision of the calculated GR activities were: Sample 1- 3.35 mU/mL, 2.7% CV Sample 2- 2.38 mU/mL, 5.6% CV Sample 3- 1.75 mU/mL, 3.9% CV Sample 4- 0.56 mU/mL, 3.7% CV Sample 5- 0.27 mU/mL, 3.6% CV Inter Assay Precision: Five native samples were diluted in Assay Buffer and run in duplicates in twenty-two assays run over multiple days by four operators. The mean and precision of the calculated GR activities were: Sample 1- 3.35 mU/mL, 5.0% CV Sample 2- 2.36 mU/mL, 12.6% CV Sample 3- 1.64 mU/mL, 6.8% CV Sample 4- 0.62 mU/mL, 10.67% CV Sample 5- 0.27 mU/mL, 10.5% CV
-
Assay cross reactivity
Human
-
Samples to be used with this kit
Serum, EDTA Plasma, Heparin Plasma, Erythrocytes, Cell Lysates
-
Maximum samples to be used with this kit
41 samples in duplicate
-
Assay duration
20 Minutes
-
Kit s sensitivity
0,009 mU/ml
-
Assay detection limit
0,156 - 5 mU/ml
-
Storage recommendations
4°C
-
Shipping recommendations
Blue Ice
-
Bibliography
1. Meister A. (1994) J Biol Chem. 269(13): 9397-400. 2. Massey V., & Williams C. (1965) J Biol Chem. 240(11): 4470-80. 3. Andersen H., Nielsen J., Nielsen F., & Grandjean P. (1997) Clin Chem. 43(4): 562-8.
-
Release date
17-Sep-2014
-
PubMed number
Refer to PubMed
-
Tested applications
No data available / currently testing
-
Tested reactivity
No data available / currently testing
-
Resources available upon request
Kit Booklet, MSDS
-
Product image link
-
Product image legend
Continuous Kinetic Assay Typical Standard Curve for Glutathione Reductase Activity Kit (Enzyme Activity Assay) StressXpress® - SKT-204. Assay Type: Direct. Detection Method: Fluorometric Assay. Assay Range: 0.156 - 5 mU/ml. | End-Point Assay Typical Standard Curve for Glutathione Reductase Activity Kit (Enzyme Activity Assay) StressXpress® - SKT-204. Assay Type: Direct. Detection Method: Fluorometric Assay. Assay Range: 0.156 - 5 mU/ml. | Chemical equation of the Glutathione Reductase reaction. The regeneration of GSH is catalyzed by Glutathione Reductase. Glutathione Reductase is an ubiquitous 100-120 kDa dimeric flavoprotein that catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione, using ß-nicotinamide dinucleotide phosphate (NADPH) as the hydrogen donor. | Linearity was determined by taking Jurkat cell lysates at 40 x 106 cells/mL diluted to 200,000 and 20,000 cells/mL and mixing in the ratios given below. The measured activities were compared to the expected values based on the ratios used. Graph of the Continuous Kinetic Assay Typical Standard Curve for the Glutathione Reductase Activity Kit StressXpress - SKT-204 | Graph of the End-Point Assay Typical Standard Curve for the Glutathione Reductase Activity Kit StressXpress - SKT-204 | Chemical Equation of the Glutathione Reductase Reaction for the Glutathione Reductase Activity Kit StressXpress - SKT-204 | Graph of the Linearity Recovery for the Glutathione Reductase Activity Kit StressXpress - SKT-204
-
Warnings
Non-hazardous
-
Country of production
Canada
-
Total weight kg
2
-
Net weight g
300
-
