Glutathione Reductase Fluorescent Kit

Katalog-Nummer OKAU00008

Size : 1plate

Marke : Aviva Systems Biology

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Telefonnummer : +1 850 650 7790

Datasheets/ManualsPrintable datasheet for OKAU00008
Product Info
ELISA Kit LinearityLinearity was determined by taking Jurkat cell lysates at 40 x 10^6 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.
Low Cell #High Cell #Expected Activity (mU/mL)Observed Activity (mU/mL)% Recovery
Mean Recovery96.5%
ELISA Kit PrincipleThe DetectX® Glutathione Reductase (GR) Fluorescent Activity Kit is designed to quantitatively measure glutathione reductase (GR) activity in a variety of samples. Please read the complete kit insert before performing this assay. 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, ThioStar®, 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 ThioStar® 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 ThioStar® 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.
ELISA Kit ReproducibilityIntra Assay Precision Five native samples were diluted in Assay Buffer and run in replicates of 16 in an assay. The mean and precision of the calculated GR activities were:
SampleGlutathione Reductase Activity (mU/mL)%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:
SampleGlutathione Reductase Activity (mU/mL)%CV
ELISA Kit Component
Black Half Area 96 Well PlateOne Plate
Glutathione Reductase Standard40 uL
ThioStar® Detection Reagent1 vial
Dry DMSO2 mL
Assay Buffer Concentrate60 mL
NADPH1 vial
NADPH Diluent5 mL
Oxidized Glutathione3 mL
Additional InformationBackground: 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. The regeneration of GSH is catalyzed by GR. GR is an ubiquitous 100-120 kDa dimeric flavoprotein that catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione, using b-nicotinamide dinucleotide phosphate (NADPH) as the hydrogen donor. 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. The general GR reaction is shown below: Glutathione Reductase GSSG 2 GSH NADPH + H+ NADP+ The most widely used procedure to measure GR is to monitor the oxidation of NADPH as a decrease in absorbance at 340 nm4. However this method suffers from the absorbance of many biological molecules at 340 nm. This DetectX® assay determines GR activity by directly measuring the amount of GSH generated from the reduction of GSSG by reacting the GSH with a non-fluorescent molecule, ThioStar®, to covalently bind the free thiol group on GSH and yield a highly fluorescent product.
::Detection Limit: 0.011 mU/mL
Reconstitution and StorageAll components of this kit should be stored at 4C until the expiration date of the kit. DMSO, when stored at 4C, will freeze. DMSO can be stored tightly capped at room temperature.
Sample TypeSerum, Plasma, RBCs and Cell Lysates
Sensitivity0.009 mU/mL
Gene Full NameGlutathione Reductase