Research use only The Protein Thiol Assay Kit is a fluorescent assay kit designed for the quantification and detection of compounds including Tris Protein thiols in common biological buffers including phosphate and citrate buffers.

This complete, ready-to-use kit includes a black 96-well plate, N-acetylcysteine standards, detection reagents, assay buffer and other components for performing the assay. Use of this kit requires a 96-well microplate reader capable of reading fluorescence emission at 510 nm (excitation at 390 nm).

Performance Characteristics
• Assay type: Fluorescent detection kit
• Sample type: Common biological buffers including Tris, phosphate and citrate buffers Liquid
• Sensitivity: 4.62 nM
• Standard curve range: 78–10,000 nM
• Reactivity: species independent

Background
Free Sulfur Alcohols play important roles in biological systems. Oxidatively modified thiol groups of cysteine residues are known to modulate the activity of an increasing number of proteins. One of the most pressing issues with this method is the accurate determination of the degree of modification of specific amino acids (e.g., cysteine residues) in complex protein samples, especially in the presence of chaotropic reagents (e.g., guanidine hydrochloride).

This thiol fluorescence detection kit allows users to accurately determine the free thiol content in a sample using a patented non-fluorescent detection reagent that converts into bright thiols upon reaction with the thiols in the sample. Fluorescent products. The thiols in the sample can be thiols produced by reactions, such as the final products of enzymatic reactions such as glutathione, or they can be the cysteine content of the protein to be tested. The assay has been tested using guanidine hydrochloride samples at concentrations up to 2M in the assay buffer provided with the kit. This allows for accurate determination of the thiol content of unfolded proteins. Although cysteine derivatives have been provided as standards that can be used to quantify free cysteine on peptides and proteins, the assay should be calibrated to a chemical standard as close as possible to the thiol being measured. For example, if the end user uses a kit to measure glutathione, the assay should be calibrated to a known, validated glutathione standard preparation.

Detection Principle
The Protein Thiol Fluorescence Detection Kit is designed for the quantitative determination of thiol groups generated or present in biological samples. Standards are provided to generate a standard curve for the assay and all samples should be read from the generated standard curve. Pipette samples and standards into black microtiter plates. After the sample or standard is mixed with the detection reagent and incubated at room temperature for 30 minutes, the fluorescent product is read at 510 nm in a fluorescent microplate reader with an excitation wavelength of 390 nm. Since biologically generated free thiols (such as glutathione and protein thiol groups) are present in different environments, the amount of thiol groups present or generated should be calibrated using appropriate standards.

Related links
Learn more about ELISA kits
Learn more about other immunoassays