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Amplite™ Fluorimetric Glutathione Assay Kit *Green Fluorescence*

GSH dose responses were measured in a solid black 96-well plate with Amplite™ Fluorimetric Glutathione Assay Kit using a NOVOstar microplate reader (BMG Labtech).
GSH dose responses were measured in a solid black 96-well plate with Amplite™ Fluorimetric Glutathione Assay Kit using a NOVOstar microplate reader (BMG Labtech).
Ordering information
Price ()
Catalog Number10055
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


The monitoring of reduced and oxidized glutathione (GSH) in biological samples is essential for evaluating the redox and detoxification status of cells and tissues in relation to the protective role of glutathione against oxidative and free-radical-mediated cell injury. Cysteine metabolism disorders include cystinosis, an autosomal recessive disease produced by a defect in lysosomal transport, and cystinuria, a common heritable disorder of amino acid transport. Cysteine is unique among the amino acids found in proteins. There are a few reagents or assay kits available for quantitating thiols in biological systems. However, all the commercial kits either lack sensitivity or have tedious protocols. Our Amplite™ Fluorimetric Glutathione Qutitation Kit provides an ultrasensitive fluorimetric assay to quantitate GSH in sample. The kit uses a proprietary non-fluorescent dye that becomes strongly fluorescent upon reacting with thiol. The kit provides a sensitive, one-step fluorimetric method to detect as little as 1 picomole of cysteine or GSH in a 100 µL assay volume. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read using a fluorescence microplate reader.

Platform


Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black

Components


Component A: Thiolite™ Green1 vial
Component B: Assay Buffer1 bottle (25 mL)
Component C: GSH Standard1 vial (62 µg)
Component D: DMSO1 vial (200 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare GSH working solution (50 µL)
  2. Add GSH standards or test samples (50 µL)
  3. Incubate at RT for 10 to 60 minutes
  4. Monitor the fluorescence increase at Ex/Em = 490/525 nm (Cutoff = 515 nm)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

 

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. GSH standard solution (1 mM):
Add 200 µL of Assay Buffer (Component B) into the vial of GSH Standard (Component C) to make 1 mM (1 nmol/µL) GSH standard solution.

2. Thiolite™ Green stock solution (100X):
Add 100 µL of DMSO (Component D) into the vial of Thiolite™ Green (Component A) to make 100X Thiolite™ Green stock solution.

PREPARATION OF STANDARD SOLUTION

GSH standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/10055

Add 30 μL of 1 mM (1 nmol/µL) GSH standard solution to 970 μL of Assay Buffer (Component B) to generate 30 μM (30 pmol/µL) GSH standard solution. Take 30 μM (30 pmol/µL) GSH standard solution and perform 1:3 serial dilutions to get serially diluted GSH standards (SD7-SD1) with Assay Buffer (Component B). Note: Diluted GSH standard solution is unstable. Use within 4 hours.


PREPARATION OF WORKING SOLUTION

Add 50 μL of 100X Thiolite™ Green stock solution into 5 mL of Assay Buffer (Component B) and mix well to make GSH working solution. 

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of GSH standards and test samples in a solid black 96-well microplate. SD = GSH Standards (SD1 - SD7, 0.01  to 10 µM); BL=Blank Control; TS=Test Samples

BL BL TS TS
SD1 SD1 ... ...
SD2 SD2 ... ...
SD3 SD3    
SD4 SD4    
SD5 SD5    
SD6 SD6    
SD7 SD7    

Table 2. Reagent composition for each well.

Well Volume Reagent
SD1-SD7 50 µL Serial Dilutions (0.01 to 10 µM)
BL 50 µL Assay Buffer
TS 50 µL Test Sample
  1. Prepare GSH standards (SD), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.  Note: Treat cells or tissue samples as desired.

  2. Add 50 µL of GSH working solution into each well of GSH standard, blank control, and test samples to make the total assay volume 100 µL/well. For a 384-well plate, add 25 µL of GSH working solution into each well instead, for total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 10 to 60 minutes, protected from light.

  4. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 490/525 nm (Cutoff = 515 nm). 

Citations


View all 2 citations: Citation Explorer
Xylitol acts as an anticancer monosaccharide to induce selective cancer death via regulation of the glutathione level
Authors: Tomonobu, Nahoko and Komalasari, Ni Luh Gede Yoni and Sumardika, I Wayan and Jiang, Fan and Chen, Youyi and Yamamoto, Ken-ichi and Kinoshita, Rie and Murata, Hitoshi and Inoue, Yusuke and Sakaguchi, Masakiyo
Journal: Chemico-Biological Interactions (2020): 109085
Comparison of different methods to study effects of silver nanoparticles on the pro-and antioxidant status of human keratinocytes and fibroblasts
Authors: Ahlberg, Sebastian and Rancan, Fiorenza and Epple, Matthias and Loza, Kateryna and Höppe, David and Lademann, Jürgen and Vogt, Annika and Kleuser, Burkhard and Gerecke, Christian and Meinke, Martina C
Journal: Methods (2016)

References


View all 131 references: Citation Explorer
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A highly sensitive fluorescence probe for fast thiol-quantification assay of glutathione reductase
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Journal: Angew Chem Int Ed Engl (2009): 4034
A 1,536-well-based kinetic HTS assay for inhibitors of Schistosoma mansoni thioredoxin glutathione reductase
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A high-throughput reporter gene assay to prove the ability of natural compounds to modulate glutathione peroxidase, superoxide dismutase and catalase gene promoters in V79 cells
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Journal: J Chromatogr A (2007): 130
A high pressure liquid chromatography-based assay for glutathione-S-transferase class distinction assay
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Journal: J Biochem Biophys Methods (2007): 761
Fluorometric assay for the determination of glutathione reductase activity
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Relationship between the expression of P-glycoprotein, glutathione S-transferase-pi and thymidylate synthase proteins and adenosine triphosphate tumor chemosensitivity assay in cervical cancer
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Journal: Zhonghua Fu Chan Ke Za Zhi (2007): 201
Colorimetric coupled enzyme assay for gamma-glutamyltransferase activity using glutathione as substrate
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Correlation between formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites determined by a comet assay, increased MDA, and decreased glutathione during long exposure to thinner inhalation
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Journal: Toxicol Lett (2006): 198