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Fluoro Thiol

Fluoro Thiol Cell Technology Metabolic Assays
Fluoro Thiol
Fluorescent Thiol Detection Kit - Detection of Thiols in tissue extracts /cell samples

$375.00

in stock
Product code: FLTHIO100
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Product Description

Key Benefits

  • Detection of reduced Thiols in cells or tissue extracts.
  • Sensitive Fluorescent Assay.
  • Detection of reduced Thiol levels in apoptosis, metabolism and oxidative stress.
  • Detection of reduced Thiols in Bacterial, fungal, plant cells or other samples.
  • Fluorescent 96 well Plate Reader Readout (excitation: 488nm and emission at 515-530 nm).

Assay Principle

During the normal course of metabolism, oxygen is partly reduced as electrons leak out of the electron transport chain during respiration. These partially reduced oxygen species (ROS) can react with organic substances through non-catalytic means. Furthermore, ROS can be generated via endogenous enzyme systems like plasma NADPH oxidase, cytoplasmic xanthine oxidase and organelle sources e.g., cytochrome P-450. ROS have been implicated in regulating diverse cellular functions including proliferation, defense against pathogens, intra-cellular signaling, transcriptional activation and apoptosis. Elevation of ROS beyond the buffering capacity of the cell can lead to oxidative stress. Elevated ROS levels can lead to damage of DNA/RNA, proteins and lipids which may lead to apoptosis. Cells have developed several mechanisms to counter act elevated ROS levels such as a thiol reducing buffer composed of cellular thiol levels (glutathione and thioredoxion) for the maintenance of the reduction-oxidation (redox) state of the cell, and enzymes to remove ROS (catalase, superoxide dismutase and glutathione peroxidase) (1-2).

Figure 1. Detection of glutathione (GSH) utilizing the Fluoro Thiol kit.

Figure 2. Jurkat cells were incubated with 1 mM staurosporine (4) for 3 hours. After which thiol levels were quantified using Cell Technology’s Fluoro Thiol kit. The graph represents approximately 1 x 10 4 cells per reaction (n=3).

Reaction

Cell Technology’s Fluoro- Thiol kit detects Thiol levels in cells and tissue extracts. Hatsuo Maeda and co workers have developed a quenched dye as a fluorescent specific probe for general thiol detection (3). The reaction scheme is outlined below.

Thiol (reduced) + non-fluorescent Dye ——-> fluorescent analog
excitation: 488nm and emission at 515-530 nm

References

  • Gamaley IA and Klyubin IV (1999) Roles of reactive oxygen species: Signaling and regulation of cellular functions. Int Rev Cytol 188:203–238.
  • Nakamura H, Nakamura K and Yodoi J (1997) Redox regulation of cellular activa-tion. Annu Rev Immunol 15:351–369.
  • 2,4-Dinitrobenzenesulfonyl Fluoresceins as Fluorescent Alternatives to Ellman s Reagent in Thiol-Quantification Enzyme Assays*. Hatsuo Maeda,*Hiromi Matsuno,Mai Ushida, Kohei Katayama,Kanako Saeki,and Norio Itoh. Angew.Chem.Int.Ed.2005 ,44 ,2922 –2925
  • Marchetti,P.,et al .,Redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function.Eur.J.Immunol.,27 ,289-296 (1997).

Kit contents and Long Term storage

ReagentPart#Temperature
Dye, 1 Vial DriedPart# 4021Store at -20C
Lysis Buffer, 25 mLPart# 3053Store at 2-8C
GSH Standard, 3 Vials DriedPart# 7015Store at 2-8C
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