Microtiter plate assay for superoxide dismutase using MTT reduction by superoxide.
A simple microtiter plate based colorimetric assay for superoxide dismutase is described. The method, involves generation of superoxide by pyrogallol autoxidation and the inhibition of superoxide dependent reduction of the tetrazolium dye MTT [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide] to its formazan, measured at 570 nm. The reaction was terminated by the addition of dimethyl sulfoxide (DMSO) which also helps to solubilize the formazan formed and the colour evolved was stable for many hours. The method was compared with other known methods to measure the activity of purified erythrocyte Cu,ZnSOD and superoxide dismutase activity from various rat tissues. This procedure involves inexpensive reagents, allows a rapid and sensitive measurement of SOD activity and the microtiter plate assay is suitable for use with large number of samples.
Madesh
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M
Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College Hospital, Vellore, India.
Balasubramanian
K A
KA
D000818:Animals; D002417:Cattle; D003124:Colorimetry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology; D005562:Formazans / Q000032:analysis; D007700:Kinetics; D011748:Pyrogallol / Q000378:metabolism; D051381:Rats; D013482:Superoxide Dismutase / Q000032:analysis*; D013481:Superoxides / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
D004791:Enzyme Inhibitors; D005562:Formazans; D013778:Tetrazolium Salts; D013844:Thiazoles; D011748:Pyrogallol; D013481:Superoxides; D013482:Superoxide Dismutase; C022616:thiazolyl blue