Invitrogen™ Thiol Fluorescent Detection Kit

Description

The Protein Thiol Detection research-use-only kit is a fluorescent assay designed for the quantification and detection of protein thiol in common biological buffers including biological buffers including Tris, phosphate, and citrate buffers.

This complete, ready-to-use kit includes black 96-well plate(s), N-acetylcysteine 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: common biological buffers including Tris, phosphate, and citrate buffers
• Sensitivity: 4.62 nM
• Standard curve range: 78–10,000 nM
• Reactivity: species independent

Background
Free thiols in biological systems have important roles. Oxidatively modified thiol groups of cysteine residues are known to modulate the activity of a growing number of proteins. One of the most pressing problems with this approach is to accurately determine the extent of modification of specific amino acids, such as cysteine residues, in a complex protein sample, especially in the presence of chaotropic agents such as guandine hydrochloride.

This Thiol Fluorescent Detection kit allows users to accurately determine the extent of free thiol content in samples using a proprietary non-fluorescent detection reagent, that is converted to a brightly fluorescent product upon reaction with the thiol in the sample. The thiol in the sample can either be one that is generated by a reaction, such as the end product of an enzymatic reaction such as glutathione, or can be the cysteine content of the protein to be measured. This assay has been tested with samples in guanidine hydrochloride concentrations up to 2M in the Assay Buffer supplied in the kit. This allows the thiol content of unfolded proteins to be accurately determined. Although a cysteine derivative has been provided as a standard that can be used to quantify free cysteines on peptides and proteins, the assay should be calibrated to a standard that is as close as possible chemically to the thiol being measured. For example, if the end user is measuring glutathione with the kit, then the assay should be calibrated to a known, validated glutathione standard preparation.

Assay principle
The Protein Thiol Fluorescent Detection Kit is designed to quantitatively measure thiol groups generated or present in biological samples. A standard is provided to generate a standard curve for the assay and all samples should be read off the curve generated. Samples and standards are pipetted into a black microtiter plate. After mixing the sample or standard with the detection reagent and incubating at room temperature for a 30-minute incubation, the fluorescent product is read at 510 nm in a fluorescent plate reader with excitation at 390 nm. Since biologically generated free thiols, such as glutathione, and protein thiol groups, exist in different environments, the amount of thiol present or generated should be calibrated using a suitable standard.