Invitrogen™ Glutathione Fluorescent Detection Kit

Descripción

The Glutathione Detection research-use-only kit is a fluorescent assay designed for the quantification and detection of glutathione in whole blood, serum, plasma, erythrocytes, urine, cell lysates and tissue samples.

This complete, ready-to-use kit includes black 96-well plate(s), glutathione standard, detection reagent, assay buffer, and other components to perform the assay. A 96-well microplate reader capable of reading fluorescent emission at 510 nm, with excitation at 390 nm, is required for use of this kit.

Performance characteristics
• Assay type: fluorescent detection kit
• Sample types: whole blood, serum, plasma, erythrocytes, urine, cell lysates, and tissue samples
• Sensitivity: 45 nM
• Standard curve range: 0.2–25 μM
• Reactivity: species independent

Background
Glutathione (GSH) is the highest concentration non-protein thiol in mammalian cells and is present in concentrations of 0.5–10 mM. 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 reductant. The oxidized GSH dimer, GSSG, is formed from GSH and peroxide by the GP reaction.

An important role of GSSG in the NFkappaB activating signal cascade is suggested by the facts that the potent NFkB inducer, tetradecanoyl phorbol acetate, increases intracellular GSSG levels and GSSG/GSH ratios. 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.

Assay principle
The Glutathione Fluorescent Detection Kit is designed to quantitatively measure glutathione (GSH), and oxidized glutathione (GSSG) present in a variety of samples. The kit is unique in that both free and oxidized glutathione are detected in the same well in the microtiter plate. No separation or washing is required. Total glutathione is the sum of GSSG plus GSH. A GSH standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. The kit utilizes a proprietary non-fluorescent detection reagent, that will covalently bind to the free thiol group on GSH to yield a highly fluorescent product. After mixing the sample or standard with the detection reagent 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. Free glutathione, GSH, is read first after 15 minutes, followed by addition of a reaction mixture that converts all the oxidized glutathione, GSSG, into free GSH, which then reacts with the excess detection reagent to yield the signal related to Total GSH content. The total concentration of GSH generated in the sample is calculated from the generated signal.