Colon tissue samples must undergo the deproteination procedure as described on page 2 of the kit's technical bulletin.
MAK440
Glutathione GSH/GSSG Assay Kit
Sufficient for 100 colorimetric tests
Synonym(s):
GSH/GSSG Ratio Kit
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About This Item
UNSPSC Code:
12161503
NACRES:
NA.84
Pricing and availability is not currently available.
Recommended Products
Quality Level
application(s)
pharmaceutical
detection method
colorimetric
relevant disease(s)
Alzheimer′s disease; Parkinson′s disease; cancer; neurological disorders; aging/geriatric diseases
storage temp.
−20°C
General description
Glutathione (GSH), composed of the amino acids glycine, glutamic acid, and cysteine, serves as a fundamental antioxidant within the body, playing a pivotal role in cellular defense against oxidative stress caused by reactive oxygen species (ROS). As the major free thiol in cells, GSH is instrumental in various biological processes, including the detoxification of harmful substances, the removal of hydroperoxides, and the maintenance of protein sulfhydryl groups in their reduced state. GSH achieves this by directly reducing disulfide bonds in cytoplasmic proteins to cysteines, during which it is transformed into its oxidized form, glutathione disulfide (GSSG). This cycle between reduced and oxidized forms is critical for maintaining the cellular redox balance.Glutathione functions as a cofactor for several key antioxidant enzymes, enhancing their ability to neutralize free radicals. It also plays a crucial role in the regeneration of vitamins C and E, thereby contributing to their antioxidative effects. Beyond its antioxidant properties, GSH is vital for mitochondrial function and the integrity of mitochondrial DNA (mtDNA), which is essential for energy production and cellular health. Dysregulation of glutathione levels is associated with the pathogenesis of several neurodegenerative diseases such as Alzheimer′s, Parkinson′s, and Huntington′s diseases, as well as other health conditions including cystic fibrosis, immune disorders, and cardiovascular diseases. Glutathione′s role in metabolism and cellular protection is extensive, influencing cellular proliferation, apoptosis, and overall physiological stability.
Application
The Glutathione GSH/GSSG Assay Kit may be used for:
- Cancer Research
- Diabetes Research
- Neurodegenerative Disease Research
Features and Benefits
Sensitive Detection Range: Detect Glutathione levels accurately across a wide range, from 0.01 µM to 3 µM, using only 25 μL of sample in a 96-well plate setup, ensuring highly sensitive analysis with minimal sample requirement.
Simplified Process: Experience a streamlined process with the addition of only a single working reagent and a 10 minute room temperature reaction, reducing complexity and saving valuable time and effort.
Compatibility with High-Throughput Systems: Easily incorporate our kit into high-throughput handling systems, ensuring smooth and accurate processing, enhancing efficiency in your laboratory workflow.
Simplified Process: Experience a streamlined process with the addition of only a single working reagent and a 10 minute room temperature reaction, reducing complexity and saving valuable time and effort.
Compatibility with High-Throughput Systems: Easily incorporate our kit into high-throughput handling systems, ensuring smooth and accurate processing, enhancing efficiency in your laboratory workflow.
Suitability
The kit is suitable for the quantitative determination of reduced and oxidized glutathione (GSH/GSSG) in whole blood, plasma, serum, urine, tissue and cell extracts.
Principle
The Glutathione GSH/GSSG Assay Kit is designed to accurately measure total, reduced and oxidized glutathione in biological samples using an enzymatic method that utilizes Ellman′s Reagent (DTNB) and glutathione reductase (GR). DTNB reacts with reduced glutathione to form a yellow product. The rate of change in the optical density, measured at A412 nm, is directly proportional to the glutathione concentration in the sample. This kit can also be used to measure oxidized (GSSG) by using a specific protocol which first scavenges all GSH with 1-methyl-2-vinylpyridinium triflate.
Other Notes
For additional information on our range of Biochemicals, please complete this form.
Storage Class Code
10 - Combustible liquids
Flash Point(F)
188.6 °F
Flash Point(C)
87 °C
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After the extraction procedure on colon tissues, do I have to also realize the deproteination before the assay ?
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Can this kit be used to analyze plasma or serum samples? If yes, is plasma or serum better? If yes, what is the procedure to follow as the procedure is described for whole blood and cell lysate only. What is the stability of frozen plasma for glutathione?
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Serum or plasma samples can be prepared using the same method as whole blood samples. Both types of samples are suitable for testing and are equally recommended. EDTA plasma is specifically known to be compatible with the assay.
Samples of serum or plasma can be frozen for later analysis; however, it is advisable to freeze them promptly after preparation at -80°C. To minimize the risk of glutathione degradation, avoid multiple freeze-thaw cycles and aim to test the samples within one month.
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Where does the DF of the calculation come from when using Scavenger or without Scavenger? If I need to make an additional dilution do I multiply it?
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The volumes used for buffer and scavenger should not be altered from the stated volumes.
For all samples treated with Scavenger, DF = 165. For samples not treated with Scavenger, DF = 450 for whole blood and 150 for all other samples. Please refer to the technical bulletin, specifically page 4, for information on the calculation of GSH and GSSG.Helpful?
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How is the test for GSSG and total GSH performed when using dry biomass? Is only PBS used or should phosphate buffer + EDTA be added? What is the estimated sonication time?
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In this case, the use of PBS is not recommended. For preparation of a sample from the dry biomass, follow the procedures as described for preparing a cell lysate. That is, use 50 mM phosphate, pH 7, 1 mM EDTA, and 20 microliters of Scavenger for preparation of the sample for GSSG measurement, and use 50 mM phosphate, pH 7, and 1 mM EDTA (with NO scavenger) for preparation of the sample for GSH measurement. Sonication for 30-60 seconds should be sufficient, but examination of a sample under a microscope to ensure that it is completely homogenized is recommended.
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To which type of tissues this kit is suitable for? was it tested on mice brain?
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This product is not tested specifically on mouse brain tissue. However, this kit is very robust and suitable for use on all species. Please see the link below to review an article referencing the use of this item on mouse brain:
Thiosulfate sulfurtransferase deficiency promotes oxidative distress and aberrant NRF2 function in the brain
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10701433/Helpful?
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We want to buy the glutathione Assay Kit to detect the GSH/GSSG level in bile sample and we also found one publication(PMID: 23978715) used the product from your company. However, I can't find how to prepare the bile sample in the product manual.
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This kit has not been tested for use with bile. Please see the link below to review a publication that indicates suitability: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362073/
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Is there a protocol available for preparing tissue for assay with the MAK440 Glutathione GSH/GSSG Assay Kit?
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The supplier recommends the following protocol for preparing tissue for assay with the MAK440 Glutathione GSH/GSSG Assay Kit:
1. Start with 20-100 mg tissue and add 200-1000 μL of ice-cold PBS.
2. Lysis can be achieved by homogenization (10-20 passes in a Dounce homogenizer on ice) or by sonication (preferably performed in an ice-water bath). The degree of tissue lysis can be checked under a microscope.
3. Centrifuge the homogenate at 14,000 g for 10 min and transfer the clear supernatant into a clean tube.
4. It is advisable to run a pilot test of the sample at different dilutions and choose a dilution with readings in the detection range of the standard curve for further assays.
It is important to note that β-mercaptoethanol, dithiothreitol, and cysteine are known to interfere in this assay, so it is crucial to avoid the use of these compounds during sample preparation.Helpful?
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What is the procedure for preparing the 50 mM phosphate, 1 mM EDTA, pH 7.0 lysis buffer?
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1.The procedure for preparing the 50 mM phosphate, 1 mM EDTA, pH 7.0 lysis buffer involves creating the cold buffer by combining EDTA disodium salt and sodium phosphate. After making the necessary pH adjustment using phosphoric acid or a base such as a NaOH solution to reach pH 7.0, the final volume adjustment should be done. It's important to note that the provided values pertain to a 1 L buffer preparation, and adjustments can be made if a lesser quantity is required.
2.To prepare a 1 mM EDTA solution, the weight calculation involves multiplying 1 x 10^-3 M by 372.24 g/mole, resulting in 0.37224 g of EDTA per L. It's suggested to consider making a more concentrated stock (e.g., 100 mM) and then performing a 100x dilution during buffer preparation.
3.For the 50 mM phosphate component, the sodium phosphate molecular weight of 119.98 g/mole and the phosphate molecular weight of 94.94 g/mole are used to determine the appropriate quantities. Specifically, for every 6 grams of sodium phosphate, 4.7485 g represents "phosphate." Therefore, dissolving 6 grams of sodium phosphate in 1 L yields 50 mM "phosphate."Helpful?
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Can 100,000 cells be used as a starting point in the protocol of MAK440-1KT Glutathione GSH/GSSG Assay Kit, and then be divided in half after sonication to measure GSH and GSSG separately? If a lower number of cells is used, should all the reagent volumes be proportionally reduced?
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Using 1-2 million cells typically provides GSH/GSSG readings within the detection limit of the kit. Using fewer cells will likely result in undetectable GSH/GSSG levels. while it might be possible to use fewer than 1 million cells depending on the expected GSH/GSSG levels in the sample, 100,000 cells are most likely not sufficient. Additionally, reducing the reagent volume will decrease the sensitivity of the kit.
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Hi, I'm curious if it is required to perform analysis only from freshly prepared samples or it is possible to use frozen ones, too. Is there any recommended step in which to freeze the sample to avoid the degradation of GSH? Thanks!
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Samples may be frozen prior to use with this kit. Freezing is actually part of the protocol for whole blood samples. However, reduced GSH is readily oxidized to form GSSG. This kit is capable of measuring total, reduced, and oxidized glutathione. In order to get a true profile of the sample it is best to run the assay through to completion following lysis and deproteinization. A similar product, CS0260, limits the storage of extracted samples to 10 days. Extracted sample storage has not been evaluated for the MAK440, however similar limitation would be expected. Should storage of extracted and/or deproteinized samples be required, samples should remain on ice during the assay procedure.
Please see the link below to review the protocol:
https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/179/194/mak440pis-mk.pdfHelpful?
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