Glutathione Fluorescent Detection Kit

Cat# OKAU00006-4PLATE

Size : 4plate

Marca : Aviva Systems Biology

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Telefono : +1 850 650 7790

Datasheets/ManualsPrintable datasheet for Glutathione Fluorescent Detection Kit (OKAU00006)
Product Info
ELISA Kit Detection MethodFluorometric
ELISA Kit LinearityLinearity was determined by taking human RBCs at two different concentrations and mixed in the ratios given below. The measured concentrations were compared to the expected values based on the ratios used.
High RBC SampleLow RBC SampleObserved Conc. (uM)Expected Conc. (uM)% Recovery
FreeTotalFreeTotalFreeTotal
80%20%6.2818.356.3218.2999.3%100.3%
60%40%4.7713.894.9314.3596.7%96.8%
40%60%3.3810.183.5510.4195.3%97.8%
20%80%2.046.552.166.4794.5%101.2%
Mean Recovery96.5%99.0%
ELISA Kit PrincipleThe DetectX® Glutathione Kit is designed to quantitatively measure glutathione (GSH), and oxidized glutathione (GSSG) present in a variety of samples. The kit is unique in that both free and oxidized glutathione are detected in the same well in the microtiter plate. No separation or washing is required. Total glutathione is the sum of GSSG plus GSH. Please read the complete kit insert before performing this assay. A GSH 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 to yield a highly fluorescent product. After mixing the sample or standard with ThioStar® and incubating at room temperature for 15 minutes, the fluorescent product is read at 510 nm in a fluorescent plate reader with excitation at 390 nm. The concentration of the GSH in the sample is calculated, after making a suitable correction for any dilution of the sample, using software available with most fluorescence plate readers. Free glutathione, GSH, is read first after 15 minutes, followed by addition of a reaction mixture that converts all the oxidized glutathione, GSSG, into free GSH, which then reacts with the excess ThioStar® to yield the signal related to Total GSH content. The total concentration of GSH generated 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 Two each of SSA treated human urine and whole blood samples were further diluted in 1% SSA Sample Diluent and run in replicates of 20 in an assay. The mean and precision of the calculated GSH concentrations were:
SampleGSH Conc. (uM)%CV
FreeTotalFreeTotal
11.272.304.04.7
22.003.803.14.7
38.339.774.62.7
43.894.453.02.3
Inter Assay Precision Two each of SSA treated human urine and blood samples were further diluted in 1% SSA Sample Diluent and run in duplicates in twenty assays run over multiple days by two operators. The mean and precision of the calculated GSH concentrations were:
SampleGSH Conc. (uM)%CV
FreeTotalFreeTotal
11.302.408.68.3
21.833.5714.710.0
39.3811.676.06.0
44.895.897.28.0
ELISA Kit Component
ComponentQuantity
Black Half Area 96 Well Plate1 or 5 Each
Glutathione Standard100 uL or 300 uL
ThioStar® Detection Reagent2 Plastic vials or 4 Glass vials
Dry DMSO4 mL or 20 mL
Assay Buffer Concentrate35 mL or 200 mL
NADPH Concentrate300 uL or 1.4 mL
Glutathione Reductase Concentrate300 uL or 1.4 mL
Oxidized Glutathione Control300 uL
Additional InformationBackground: Glutathione (L-γ-glutamyl-L-cysteinylglycine; GSH) is the highest concentration non-protein thiol in mammalian cells and is present in concentrations of 0.5 - 10 mM. GSH plays a key role in many biological processes, including the synthesis of proteins and DNA, the transport of amino acids, and the protection of cells against oxidation. Harmful hydrogen peroxide cellular levels are minimized by the enzyme glutathione peroxidase (GP) using GSH as a reductant. The oxidized GSH dimer, GSSG, is formed from GSH and peroxide by the GP reaction (see below). An important role of GSSG in the NFΚB activating signal cascade is suggested by the facts that the potent NFΚB inducer, tetradecanoyl phorbol acetate, increases intracellular GSSG levels and GSSG/GSH ratios. Glutathione S-transferases (GST) are an important group of enzymes that catalyze the nucleophilic addition of GSH to electrophiles. They are encoded by 5 gene families; 4 encode cytosolic GST and one encodes the microsomal form of GST. They have been implicated in a number of diseases. In asthma arachidonic acid is converted to unstable leukotriene A (LTA4). LTA4 is either hydrated to form LTB4 or it is conjugated to GSH by a GST, leukotriene C4 synthase, to form leukotriene C4. LTC4 and its derivative LTD4 are important molecules in bronchial asthma. Leukotriene C4 synthase is therefore an important therapeutic target. It has also been shown that increased expression of GSTs can lead to drug resistance. Three glutathione adducts of the drug melphalan, used to treat ovarian cancer and multiple myeloma, have been isolated from reactions involving human microsomal GSTs.
::Detection Limit: The Limit of Detection was determined as 38 nM in the Free GSH and 42 nM in the Total GSH assays
Reconstitution and Storage2°C to 8°C
Sample TypeWhole Blood, Serum, Plasma, Erythrocytes, Urine, Cell Lysates and Tissue Samples
SensitivitySensitivity was determined as 45 nM in the Free GSH and 48 nM in the Total GSH assays.
Gene Full NameGlutathione