TITLE	Assay of superoxide dismutase activity by combining electrophoresis and densitometry
AUTHOR	Ching-Nen Chen Department of Botany, National Taiwan University, Taipei, Taiwan 10764, Republic of China Shu-Mei Pan Department of Botany, National Taiwan University, Taipei, Taiwan 10764, Republic of China
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ABSTRACT	A modified technique was developed to assay superoxide dismutase (SOD) activity by combining polyacrylamide gel electrophoresis and densitometry. After electrophoresis on native polyacrylamide gels, the negative banding corresponding to the SOD activity was visualized by soaking the gels in nitroblue tetrazolium then riboflavin, and finally exposing to light. Effects of the banding of SOD activity induced by different soaking durations and light intensities were evaluated in this system. The optimal soaking duration was determined to be 15 min for each of the two soaking steps, while the optimal exposure was 30 mEm-2s-1 for 15 min. The gels were then immediately scanned with a laser densitometer, and the readings of the samples corresponding to their total SOD activity were obtained by processing the image. A standard curve was prepared with a serial dilution of partially purified SOD, whose activity was previously determined by using a spectrophotometric method. The total SOD activity of an unknown sample could be obtained by interpolating its reading to the standard curve. The activity of a single SOD isozyme of a sample could also be obtained with the same procedure. The technique was ten times more efficient than the spectrophotometric method. The interference coming from non-SOD substances in the crude extract could be removed by electrophoresis. The standard deviations of the SOD activity of the crude extracts from rice seedlings, papaya, and tobacco leaves measured with the technique were less than 9%, 7%, and 8% (for each n = 6, on 6 gels), respectively.
KEYWORD	Densitometry; SOD isozyme; Superoxide dismutase;
ARTICLE INFO	Botanical Bulletin of Academia Sinica, Volume 37 Number 2 April 1996, page 107-111, 5 pages
PUBLISHER	Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China