Can anyone explain a detail method to determine superoxidase dismutase (SOD), peroxidase and catalase activity from roots?
Thanks!!
I always found the Amplex Red kit good for testing peroxidase activity (https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Fmp22188.pdf).
You probably want to flash freeze a known weight of the sample with liquid nitrogen and grind them with a mortar and pestle before solvating them in a fixed volume of distilled water.
H
Maybe this article will be useful to you.
doi: 10.1038/nprot.2009.197. Epub 2009 Dec 17.. 2010 Jan;5(1):51-66.Nat Protoc
Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue
Christine J Weydert, Joseph J Cullen
PMID: 20057381. PMCID: PMC2830880. DOI: 10.1038/nprot.2009.197
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The analysis of SOD, POD, and CAT were separately performed for leaves and roots.
The freshly cut root and leaf samples were wrapped in aluminum foil and frozen in liquid nitrogen. The frozen samples were crushed with the help of a pestle and mortar, and about 0.5 g of the powdered sample were mixed with 5 mL 50 mM of sodium phosphate buffer (pH 7.8); the mixture was 12,000* g for 15 min and the supernatant was collected for SOD, POD, and CAT estimation.
The SOD activity was determined according to the procedure of Han et al. In detail, 2.8 mL 50 mM sodium phosphate buffer (pH 7.8), 130 Mm methionine, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.75 mM Nitro blue tetrazolium (NBT), 20 uM riboflavin, and 50 uL of the enzyme extract (supernatant) or 50 uL distill water (blank) were used. Finally, the riboflavin was added to the tube mixture to start the reaction. The mixture was vortexed and incubated under constant light (4000 l*) for 20 min. The reaction was stopped in the dark, and absorbance was measured at 560 nm. SOD activity was expressed as nanogram/mL.
For peroxidase (POD), the reaction mixture contained 2.5 mL of 50 mM sodium phosphate buffer (pH 7), 1 mL of 1% H2O2, 1 mL of 1% guaiacol, and the enzyme extract (supernatant). The changes in absorbance were measured at 420 nm for 1 min. The activity was expressed in units, where one unit represented an increase in absorbance of one in one minute.
The CAT activity was measured according to Aebi et al. In detail, we mixed 100 uL enzyme source into 6 mL 50 mM sodium phosphate buffer (pH 7.8) and 20 mM H2O2. The enzyme activity was recorded at 10 s intervals for 4 min at 240 nm. The optimal activity was measured and defined as the decline of absorbance at 240 nm 0.1 unit per minute. The CAT was expressed as U g-1 FW min-1.
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