Amplite® Fluorimetric Total Thiol Quantitation Assay Kit *Green Fluorescence*
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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International | See distributors |
Bulk request | Inquire |
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Shipping | Standard overnight for United States, inquire for international |
Spectral properties
Excitation (nm) | 505 |
Emission (nm) | 524 |
Storage, safety and handling
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | SDSProtocol |
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
Excitation | 490 nm |
Emission | 525 nm |
Cutoff | 515 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol Summary
- Prepare GSH working solution (50 µL)
- Add GSH standards or test samples (50 µL)
- Incubate at RT for 10 to 60 minutes
- Monitor the fluorescence increase at Ex/Em = 490/525 nm (Cutoff = 515 nm)
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.
Note Alternatively, if precipitation is observed while making working solution, one can make 50X stock solution using 200 µL DMSO solution.
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
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).
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
Table 2. Reagent composition for each well.
BL | BL | TS | TS |
SD1 | SD1 | ... | ... |
SD2 | SD2 | ... | ... |
SD3 | SD3 | ||
SD4 | SD4 | ||
SD5 | SD5 | ||
SD6 | SD6 | ||
SD7 | SD7 |
Well | Volume | Reagent |
SD1-SD7 | 50 µL | Serial Dilutions (0.014 to 10 µM) |
BL | 50 µL | Assay Buffer |
TS | 50 µL | Test Sample |
- 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.
- 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.
- Incubate the reaction at room temperature for 10 to 60 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 490/525 nm (Cutoff = 515 nm).
Images
Citations
View all 4 citations: Citation Explorer
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Authors: Khanna, Vidhi and Kalscheuer, Stephen and Kirtane, Ameya and Zhang, Wenqui and Panyam, Jayanth
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Journal: Cancers (2019): 491
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
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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
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Authors: Sekine, Takashi and Hirata, Tadashi and Mine, Toshiki and Fukano, Yasuo
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References
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Authors: Bayle C, Issac C, Salvayre R, Couderc F, Causse E.
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Plasma total homocysteine and other thiols analyzed by capillary electrophoresis/laser-induced fluorescence detection: comparison with two other methods
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Assays for total homocysteine and other thiols by capillary electrophoresis-laser-induced fluorescence detection. I. Preanalytical condition studies
Authors: Causse E, Issac C, Malatray P, Bayle C, Valdiguie P, Salvayre R, Couderc F.
Journal: J Chromatogr A (2000): 173
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Journal: J Chromatogr A (2000): 173
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Journal: J Chromatogr Sci (1999): 469
Quantitation of homocysteine in human plasma by capillary electrophoresis and laser-induced fluorescence detection
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Application notes
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Induction of Neurite Outgrowth in PC12 Cells
Induction of Neuritogenesis in PC12 Cells by a Pulsed Electromagnetic Field
FAQ
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