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Amplite® Fluorimetric Total Thiol Quantitation Assay Kit *Green Fluorescence*

GSH and cysteine dose responses were measured on a solid black 96-well plate with Amplite® Fluorimetric Thiol Quantitation Assay Kit using a NOVOstar microplate reader (BMG Labtech).
GSH and cysteine dose responses were measured on a solid black 96-well plate with Amplite® Fluorimetric Thiol Quantitation Assay Kit using a NOVOstar microplate reader (BMG Labtech).
GSH and cysteine dose responses were measured on a solid black 96-well plate with Amplite® Fluorimetric Thiol Quantitation Assay Kit using a NOVOstar microplate reader (BMG Labtech).
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Spectral properties
Excitation (nm)505
Emission (nm)524
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Excitation (nm)
505
Emission (nm)
524
The detection and measurement of free thiol (such as free cysteine, glutathione and cysteine residues in proteins) is one of the essential tasks for investigating biological processes and events in many biological systems. The monitoring of reduced (GSH) and oxidized glutathione 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. Disorders of cysteine metabolism include cystinosis, an autosomal recessive disease produced by a defect in lysosomal transport, and cystinuria, a common heritable disorder of amino acid transport. There are 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 Total Thiol Qutitation Kit provides an ultrasensitive fluorimetric assay for quantitating thiols that exist either in a small molecule. 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 (10 nM in concentration). The assay is rapid and robust. It can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation. For rapid quantifying thiol groups in a protein, we recommend you use our kit #5529 that is optimized for quantifying protein thiols.

Platform


Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black

Components


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

PREPARATION OF STOCK SOLUTIONS

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 ddH2O 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.
Note     Alternatively, if precipitation is observed while making working solution, one can make 50X stock solution using 200 µL DMSO solution.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/5524


GSH standard
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.
Note     This GSH working solution is enough for one 96-well plate. It is unstable at room temperature, and should be used promptly within 2 hours. Avoid exposure to light.
Note     Alternatively, one can make GSH working solution by adding 100X Thiolite™ Green stock solution with Assay Buffer (Component B) proportionally.
Note     If precipitation is observed with this protocol, then one can make 50X dilution (See note in stock solution (2)) and use 100 µL of 50X Thiolite™ Green stock solution into 5 mL of Assay Buffer (Component B).

SAMPLE EXPERIMENTAL PROTOCOL

Table 1.Layout of GSH standards and test samples in a solid black 96-well microplate. SD = GSH Standards (SD1 - SD7, 0.014 to 10 µM); BL=Blank Control; TS=Test Samples
BLBLTSTS
SD1SD1......
SD2SD2......
SD3SD3
SD4SD4
SD5SD5
SD6SD6
SD7SD7
Table 2. Reagent composition for each well.
WellVolumeReagent
SD1-SD750 µLSerial Dilutions (0.014 to 10 µM)
BL50 µLAssay Buffer
TS50 µLTest 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 to 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). 

Spectrum


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spectrum

Spectral properties

Excitation (nm)505
Emission (nm)524

Images


Citations


View all 4 citations: Citation Explorer
Perlecan-targeted nanoparticles for drug delivery to triple-negative breast cancer
Authors: Khanna, Vidhi and Kalscheuer, Stephen and Kirtane, Ameya and Zhang, Wenqui and Panyam, Jayanth
Journal: Future Drug Discovery (2019)
Improving Payload Capacity and Anti-Tumor Efficacy of Mesenchymal Stem Cells Using TAT Peptide Functionalized Polymeric Nanoparticles
Authors: Moku, Gopikrishna and Layek, Buddhadev and Trautman, Lana and Putnam, Samuel and Panyam, Jayanth and Prabha, Swayam
Journal: Cancers (2019): 491
Antibody Conjugated Nanoparticles for Targeting Metastatic Triple Negative Breast Cancer
Authors: Khanna, Vidhi Devendra
Journal: (2016)
Activation of transcription factors in human bronchial epithelial cells exposed to aqueous extracts of mainstream cigarette smoke in vitro
Authors: Sekine, Takashi and Hirata, Tadashi and Mine, Toshiki and Fukano, Yasuo
Journal: Toxicology mechanisms and methods (2016): 22--31

References


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