Fluorometric quantitation of cellular and nonprotein thiols |
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| Authors: | F C Ayers G L Warner K L Smith D A Lawrence |
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| Affiliation: | 1. Food and Nutraceutical Laboratory, Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur 176061, Himachal Pradesh, India;2. Regulatory Research Centre, CSIR-Institute of Himalayan Bioresource Technology, Palampur 176061, Himachal Pradesh, India;3. Academy of Scientific and Innovative Research (AcSIR), CSIR-Institute of Himalayan Bioresource Technology, Palampur 176061, Himachal Pradesh, India;1. Burnet Institute, Melbourne, Victoria, Australia;2. Queens College of City University of New York, Flushing, USA;3. Deakin University, Victoria, Australia;4. CSIRO AAHL, Victoria, Australia;3. Key Laboratory of Resources Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi''an, Shaanxi, China 710069;4. Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina 27710;5. Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710;1. Microbial Molecular Genetics Laboratory, Institute of Biosciences and Applications, National Center for Scientific Research "Demokritos,” 15341 Agia Paraskevi, Greece;2. Department of Biology, National and Kapodistrian University of Athens, Panepistimioupolis, 15784 Athens, Greece;3. Bioanalytical Chemistry Section, Department of Biology/Chemistry, Osnabrück University, 49076 Osnabrück, Germany;4. Biotechnology Division, Systems Biology Center, Biomedical Research Foundation of the Academy of Athens, 11527 Athens, Greece;5. Institute for Bioinnovation, Biomedical Sciences Research Center “Alexander Fleming,” 16672 Vari, Greece;6. Molecular Physiology of the Cell Laboratory, Université Libre de Bruxelles (ULB), IBMM, 6041 Gosselies, Belgium;7. Center for Cellular Nanoanalytic Osnabrück (CellNanOs), Osnabrück University, 49076 Osnabrück, Germany |
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| Abstract: | A microfluorometric assay for thiols has been developed using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM). The technique may be used to quantitate either cellular or plasma thiols over a range of 0.01 to 3.0 nmol and may be used with as few as 1-3 X 10(5) cells giving highly proportional and reproducible results. Values for nonprotein thiols obtained with this assay agree well with previous reports on glutathione (GSH) levels for both lymphocytes and plasma. Readings are determined with the aid of an automated fluorescence microplate reader which allows up to 96 samples, including standards, to be read at the same time. Cellular thiols accessible after lysis were also quantitated before and after treatment of intact cells with various thiol-reactive chemicals. Interestingly, HgCl2, bromoethanesulfonic acid, and N-ethylmaleimide differentially modified protein and nonprotein thiol levels. |
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