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Cat. No.: FSAKD-200 (for 100-200T)

Cat. No.: FSAKD-1k (for 500-1000T)

Description

Free Sulfhydryl Assay Kit with DTNB or Free Thiol Assay Kit with DTNB, also known as the Exposed/Surface Free Sulfhydryl Groups Assay Kit or Free Thiol Quantification Assay Kit, is an efficient and convenient assay kit for detecting exposed or surface free sulfhydryl groups in cell culture supernatants, serum, plasma, cell and tissue lysates. This assay kit utilizes absorbance-based detection.

Sulfhydryl groups or thiols, also known as mercaptans or thiols, have the chemical formula -SH. Thiols are highly reactive and widely present functional groups in biological systems. They are found in most proteins and some low-molecular-weight substances such as glutathione (GSH), CoA, fatty acids, DTT, mercaptoacetate, and free cysteine, among others. The free sulfhydryl groups in proteins can be categorized into exposed/surface free sulfhydryl groups and buried free sulfhydryl groups within the higher-order protein structure. The sum of these two categories constitutes the total free sulfhydryl groups or unbound thiols. Typically, when referring to free sulfhydryl groups in proteins, it means the exposed free sulfhydryl groups, also known as active thiols, primarily present in cysteine residues. These active thiols play a crucial role in the protein structure and certain redox reactions in the biological system. The formation of disulfide bonds (-S-S-) through the dehydrogenation of adjacent thiols in proteins facilitates the connection of neighboring peptides. This assay kit can be used to detect exposed or surface free sulfhydryl groups in both protein and non-protein samples.

The main types of thiols in the biological system are glutathione thiols and protein thiols. Glutathione thiols primarily participate in repairing oxidatively damaged proteins and eliminating reactive oxygen species. Protein thiols, on the other hand, play a role in maintaining the conformation of proteins through the formation of disulfide bonds. An increase in detected free sulfhydryl groups in proteins indicates a breakage of disulfide bonds, suggesting a change in protein structure. Traditional methods for thiol determination include high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and liquid chromatography-mass spectrometry (LC-MS). While these methods have advantages in sensitivity and accuracy, they are relatively complex, require sophisticated instruments, are time-consuming, and cannot simultaneously analyze multiple samples.

This assay kit employs the classic DTNB-based method for quantifying exposed free sulfhydryl groups. 5,5'-Dithio-bis-(2-nitrobenzoic acid) (DTNB), also known as Ellman's reagent, is a water-soluble reagent that interacts with thiols. It is used to determine the content of free sulfhydryl groups in tissues, proteins, and peptides. The basic reaction principle is as follows: Free sulfhydryl groups (R-SH) react with DTNB to generate a yellow detectable product, 2-nitro-5-thiobenzoic acid (TNB), and a mixed disulfide (R-S-TNB). TNB has a strong absorption peak at 412 nm, and its absorbance can be measured using a spectrophotometer. By using glutathione as a standard, the thiol content in the sample can be calculated.

Components

• Assay Buffer
• GSH
• DTNB 

Features

• This assay kit offers simple sample handling and rapid detection. It can directly detect cell culture supernatant, serum, and plasma samples. For cell and tissue samples, homogenization and extraction are required, and the resulting supernatant is used for detection. The entire assay process can be completed in less than half an hour.
• The Assay Buffer in this kit has been optimized to chelate divalent metal ions capable of oxidizing thiols, preventing sample oxidation and ensuring that the thiol content in the sample is not underestimated.
• The kit provides DTNB and Assay Buffer necessary for the detection, along with glutathione (GSH) as a standard or positive control.

Storage

-20ºC, valid for two years. 4ºC, valid for six months. After preparing solutions of GSH and DTNB, it is recommended to aliquot and store at -20ºC, valid for at least three months.

Precautions

• This assay involves redox reactions, and therefore, all oxidizing or reducing agents can interfere with the assay. If reducing agents such as DTT, mercaptoethanol, etc., cannot be avoided in the sample, try to control the total concentration of reducing agents to be lower than the thiol concentration in the test sample. Common detergents like Triton X-100, Tween-20, etc., contain high levels of peroxides. If detergents must be used, it is advisable to use high-purity detergents labeled with lower peroxide content.
• This product is for scientific research by professionals only and should not be used for clinical diagnosis or treatment. It is not intended for use in food or drugs and should not be stored in ordinary residences.
• For your safety and health, please wear laboratory clothing and disposable gloves during operation.