Catalase activity assay kit

Protein was extracted from imbibing seeds, which were chilled for 3 days at 4 °C to stimulate germination and then placed at 22 °C under a 16-h light/8-h dark photoperiod for 24 h with different treatment. The supernatant was used as the crude extract and catalase activity was tested with the Catalase Activity Assay Kit (Beyotime, China, product No. S0051). Protein concentration was determined using the Bradford protein assay. Crude extract (5 µL) was mixed with catalase testing buffer, and 250 mM H₂O₂ was used as the substrate. Reaction time was strictly controlled and stopped with addition of stop buffer. The mixture was then added into the working color solution and incubated for at least 15 min. Absorbance at 520 nm was measured and activity was calculated. Catalase activity was indicated in units/mL or units/mg. One unit of catalase activity is defined as the quantity of enzyme catalyzing the decomposition of 1 mM H₂O₂ per minute.

SOD activity was studied using water-soluble tetrazolium salt (WST-1) method with the commercially available kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacture's protocol. Briefly, after treatment, the cell lysate was centrifuged and then protein level was quantified. Cell lysates (50 μg protein/ml) were subjected for assay. CAT activity was measured using catalase activity assay kit (Beyotime Biotechnology) as per the manufacturers instructions. The developed red color product, N-4-antipyryl-3-chloro-5-sulfonate-p-benzoquinonemonoimine, was absorbed maximally at 520 nm.