Glutathione Fluorescent 384-Well Detection Kit

Catalog No. ABIN2815087

Product Details

Target: Glutathione

Detection Method: Fluorometric

Host: Human

Application: Detection (D)

Purpose: The DetectX® Glutathione kit is designed to quantitatively measure glutathione (GSH), and oxidized glutathione (GSSG) present in a variety of samples.

Brand: DetectX®

Sample Type: Blood, Serum, Plasma, Erythrocyte Lysates, Urine, Cell Lysate, Tissue Samples

Components: Black 384 Well plate Kit 2 Each
Reduced Glutathione Standard Glutathione at 250 μM in a special stabilizing solution. 100 μL
Oxidized Glutathione Standard Oxidized Glutathione at 250 μM in a special stabilizing solution 350 μL
ThioStar® detection Reagent ThioStar thiol detection substrate stored in a ziploc pouch with desiccant. Reconstitute with dry DMSO. 1 Bottle dry dmSo Dry Dimethyl sulfoxide solvent over molecular sieves. May be stored at room temperature. 5 mL
Assay Buffer Concentrate A buffer containing detergents and stabilizers. A 2X concentrate that should be diluted with deionized or distilled water. 200 mL
NADHP Concentrate Reduced ß-nicotinamide adenine dinucleotide 2'-phosphate (NADPH) as a stable solution. 500 μL
Glutathione Reductase Concentrate Glutathione Reductase (GR) as a stable solution. 500 μL

Material not included: Distilled or deionized water Repeater pipet with disposable tips capable of dispensing 5 μL.
Aqueous 5-sulfo-salicylic acid dihydrate (SSA, Sigma-Aldrich ) solution at 5 % weight/volume (1g of SSA per 20 mL of water) for treating samples to remove protein. 2-vinylpyridine (Sigma ) and ethanol (Sigma ).
Fluorescence 384 well plate reader capable of reading fluorescent emission at 510 nm, with excitation at 390 nm.
Please contact your plate reader manufacturer for suitable filter sets.
Set plate parameters for a 384-well Corning Costar 3676 plate.
See: www.arborassays.com/ resources/#general-info for plate dimension data.
The sensitivity of fluorescent assays is dependant on the capabilities of the plate reader.
If your plate reader has adjustable gain you can modify the signals obtained from the assay by increasing or decreasing the gain settings, by changing the aperture settings for monochromator based readers, or by changing the band pass width of the emission and/or excitation filters on some readers.
Please review the plate reader manual for details.
Signals expressed by plate readers are Relative Fluorescent units (RFu) and the values given in the insert were obtained on our plate readers. the RFu numbers you obtain may be different from these, but the assay results should be similar.
Software for converting raw relative fluorescent unit (FLU) readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.

Application Details

Application Notes: GSH is identical across species and we expect this kit may measure GSH from sources other than human.
The end user should evaluate recoveries of GSH in samples from other species being tested.
If samples need to be stored after collection, we recommend storing them at -70 °C or lower, preferably after being frozen in liquid nitrogen.
This assay has been validated for human whole blood, serum, EDTA and heparin plasma, urine, and isolated erythrocytes.
Most cell lysates and tissue homogenates should also be compatible.
Samples containing visible particulate should be centrifuged prior to using.
All samples will be deproteinized with 5 % SSA (see page 6 for preparation), please see sample specific information below for details.
This treatment removes any protein thiols present in the samples and also slows oxidation of free GSH.

Protocol: The kit is unique in that free and oxidized glutathione are detected in a 384-well 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.
GSH and GSSG standards are provided to generate standard curves 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.
Oxidized glutathione, GSSG, is measured after blocking any GSH in the sample by treatment with 2-Vinylpyridine.
GSSG is converted into free GSH using our stable, liquid formulations of NADPH and Glutathione Reductase.
The GSH formed then reacts with ThioStar® to yield the signal related to Oxidized GSH content.
The total concentration of GSH generated in the sample is calculated from the measured Reduced and Oxidized GSH.
We have provided special 384 well plates 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.

Sample Preparation:

reduced and oxidized Glutathione Measurements reduced Glutathione is measured by taking the solutions prepared and diluted in Sample Diluent without any further treatment. To measure oxidized Glutathione in samples, reduced Glutathione (GSH) in the sample must be blocked by treatment with 2-vinylpyridine, (2VP). A solution of 27 μL of 2VP is added to 98 μL of ethanol in a fume hood. SSA treated samples are then treated with 1 μL of the ethanolic 2VP solution for every 50 μL of sample and incubating for 1 hour at room temperature. 2VP treated samples must be read off a standard curve made with 2VP-treated standards. use all samples within 2 hours of dilution. All samples and standards must be in Sample Diluent before starting the assay. All samples must be treated with the SSA solution prepared on page 6. All of the SSA treated centrifuged supernatants must have their SSA concentration brought down to 1 % SSA by dilution with Assay Buffer. Further dilutions of the sample, using Sample Diluent (see page 9 for preparation), may be necessary to allow the GSH concentration to be measurement in the assay. Detailed instructions follow. Whole Blood, eDtA or heparin Plasma, or urine Thoroughly mix sample with an equal volume of cold 5 % SSA. Incubate for 10 minutes at 4 °C. Centrifuge at 14,000 rpm for 10 minutes at 4 °C. Collect the supernatant. If the supernatent contains particulates, re-centrifuge the supernatant for 15 minutes and collect the clarified second supernatant. Samples can be stored in aliquots at ≥ -70 °C or analyzed immediately. At this point the SSA concentration will be 2.5 %. The supernatant must be diluted 1:2.5 with Assay Buffer by mixing one part with 1.5 parts of Assay Buffer. The SSA concentration will be 1 %. The sample will have been diluted 1:5 at this point. All final dilutions are to be made in Sample Diluent. Treated Whole Blood must be further diluted at least 1:20 for a recommended final dilution of ≥ 1:100. For Treated Plasma and Treated Urine a final dilution of ≥ 1:5 is recommended, but further dilutions in Sample Diluent may be necessary.

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.
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.

Restrictions: For Research Use only

Handling

Precaution of Use: As with all such products, this kit should only be used by qualified personnel who have had laboratory safety instruction.
Sulfosalicylic acid is a strong acid solution and should be treated like any other laboratory acid. 2Vp is toXiC and may cause burns. 2Vp solutions should be prepared in a fume hood.
Use immediately and discard remaining unused solutions by mixing with copious amounts of water.
Dimethyl sulfoxide is a powerful aprotic organic solvent that has been shown to enhance the rate of skin absorption of skin-permeable substances.
Wear protective gloves when using the solvent.
NOTE: DMSO can dissolve certain plastics used in troughs, etc. thioStar® thiol detection Reagent should be stored at 4°C in the desiccated pouch.
Allow desiccated pouch to warm to room temperature prior to opening. thioStar will react with strong nucleophiles.
Buffers containing the preservatives sodium azide, proclin™ and Kathon™ will react with the substrate.
Reconstituted ThioStar in DMSO stored at 4°C in the supplied desiccated pouch can be used up to 2 months later.
The background on the reconstituted ThioStar will increase slowly over time but the increase will not affect the assay results obtained.

Storage: 4 °C

Storage Comment: All components of this kit should be stored at 4°C until the expiration date of the kit. DMSO, when stored at 4°C, will freeze. Can be stored tightly capped at room temperature.

References

Engström-Öst, Glippa, Feely, Kanerva, Keister, Alin, Carter, McLaskey, Vuori, Bednaršek: "Eco-physiological responses of copepods and pteropods to ocean warming and acidification." in: Scientific reports, Vol. 9, Issue 1, pp. 4748, (2019) (PubMed).

Al-Saleh, Coskun, Al-Doush, Al-Rajudi, Al-Rouqi, Abduljabbar, Al-Hassan: "Exposure to phthalates in couples undergoing in vitro fertilization treatment and its association with oxidative stress and DNA damage." in: Environmental research, Vol. 169, pp. 396-408, (2019) (PubMed).

Target Details

Target: Glutathione

Abstract: Glutathione Products

Synonyms: GT Kit, ILBP Kit, ILLBP Kit, PIP Kit, I-15P Kit, I-BABP Kit, ILBP3 Kit, Illbp Kit, I-BALB Kit, I-BAP Kit, fatty acid binding protein 6 Kit, fatty acid binding protein 6, ileal (gastrotropin) Kit, FABP6 Kit, Fabp6 Kit

Target Type: Chemical

Background: 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 mM1. 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 reductant2. Sh nh o o 2 h o n ho n oh h o 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 ratios3. h 2 o h 2 o2 Glutathione Peroxidase GSSG GSh Glutathione reductase Glutathione S-transferase nADPh nADP+ GSh Conjugate 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 (LTA ). LTA is either hydrated 4 4 4 to form LTB or it is conjugated to GSH by a GST, leukotriene C synthase, to form leukotriene C . 4 4 4 LTC and its derivative LTD are important molecules in bronchial asthma. Leukotriene C synthase 4 4 4 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