Oxidative stress (Oxidative Stress) refers to the imbalance between oxidative and antioxidant effects in the body, which tends to oxidize, leading to inflammatory infiltration of neutrophils, increased secretion of proteases, and a large amount of oxidative intermediates. Oxidative stress is a negative effect produced by free radicals in the body and is considered to be an important factor leading to aging and disease.
The oxidation state of protein sulfhydryl groups in cells and tissues can indicate oxidative stress. However, the quantitative detection of protein sulfhydryl oxidation status in tissue samples also faces some technical obstacles, such as how to prepare samples without affecting their oxidation status, and how to detect relatively minor changes.
Recently, researchers at the University of Western Australia in Australia have developed a new fluorescence technology that can overcome the above problems and accurately determine the oxidation state of protein sulfhydryl groups. They reliably measured the overall small changes in the oxidation state of the protein's sulfhydryl groups by a fluorescence microplate reader, and then detected the oxidation state of specific proteins by SDS-PAGE.
The new technology they developed improved protein solubilization and prevented the protein in the sample from being considered oxidized. In the dissolution step, the reduced thiol is labeled with green fluorescent BODIPY FL maleimide. After removing excess BODIPY FL maleimide, the remaining oxidized protein thiol was reduced by TCEP. This newly generated reduced oxidized thiol interacts with the red fluorescent Texas Red® maleimide and removes excess dye.
Since BODIPY® FL and Texas Red® fluorophores show very little overlap in fluorescence emission, the use of these two fluorescent dyes to label proteins enables the quantitative determination of oxidative and reduced protein sulfhydryl groups in the same sample. The instrument is just a fluorescence microplate reader. By measuring oxidized and reduced protein sulfhydryl groups, the researchers were able to detect small differences in biological samples.
In addition, this dual labeling technique can also detect the sulfhydryl oxidation state of specific proteins. Through SDS-PAGE and fluorescent gel scanner, we can understand the oxidation state of specific protein thiol.
In addition to the two dyes mentioned above, Life Technologies also offers a variety of dyes for the detection of oxidative stress. In 2011, Life Tech launched the 11th edition of "Molecular Probes Fluorescent Probes and Labeling Technical Manual". This is a tool book that introduces various biomolecular labeling and detection technologies in detail. It introduces more than 3,000 technical solutions in detail and involves various biomolecular labeling and detection technologies.
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