Superoxide dismutase sod activity colorimetric assay kit

Leaf samples of the new fully expanded leaves (L1) of moth orchids with or without BL acclimation for 12 days (T0) and ML or HL exposure for two weeks were collected. A total of 100 mg of frozen leaf samples were homogenized. The SOD enzyme activity was determined using a Superoxide Dismutase (SOD) Activity Colorimetric Assay Kit (BioVision, Milpitas, CA, USA #K335-100) according to the instructions of the manufacturer. The SOD activity was calculated based on the percentage inhibition at 20 min at 37 °C. For the detection of the catalase enzyme activity, 100 mg of frozen leaf samples were homogenized and determined using a Catalase Activity Colorimetric/Fluorometric Assay Kit (BioVision, Milpitas, CA, USA #K773-100) according to the manufacturer’s instruction.

Vanilla orchid newly expanded L3 leaf samples, with or without BL-acclimation for 12 days, were exposed to ML500 or HL1000 for two weeks and then collected and stored at −80 °C freezer. A total of 100 mg of frozen leaf samples were homogenized. SOD enzyme activity was determined using a Superoxide Dismutase (SOD) Activity Colorimetric Assay Kit (BioVision #K335-100), following the instruction of the manufacturer. SOD enzyme activity was calculated based on the percentage inhibition after 20 min of incubation at 37 °C. For detection of catalase (CAT) enzyme activity, 100 mg frozen leaf samples were homogenized and determined using a Catalase Activity Colorimetric/ Fluorometric Assay Kit (BioVision, #K773-100), as described previously [22 (link)].
Vanilla orchid L3 leaves, with or without BL-acclimation for 12 days, which had then been exposed to moderate light (ML500) or high light (HL1000) irradiation for two weeks, were collected. A total of 100 mg of leaf samples were homogenized, 500 μL of 1 × PBS buffer was added, and then the mixture was incubated for 30 min at 37 °C. Then, glucose and sucrose concentrations were determined using a Glucose and Sucrose Colorimetric/Fluorometric Assay Kit (Sigma #MAK013), following the instructions provided by the manufacturer.

The Sod activity was measured using a Superoxide Dismutase (SOD) Activity Colorimetric Assay Kit (BioVision, Milpitas, CA) according to the manufacturer’s instructions. Protein extraction was performed as described above in an extraction buffer containing 0.1 M Tris/HCl (pH 7.4), 0.5% Triton X-100, 5 mM β-mercaptoethanol, and 0.1 mg/mL phenylmethylsulfonyl fluoride. Proteins (2 µg in 250 µL solution) were mixed with 200 µL WST Solution and 20 µL SOD Enzyme Solution (BioVision) and incubated at 37 °C for 20 min. The absorbance at 450 nm was measured spectrophotometrically. The relative enzyme activity of Sod was calculated by the activity in the sfp1Δ/sfp1Δ mutant divided by that in the wild-type strain.
Catalase enzyme activity was determined using a spectrophotometric method as previously described [28 (link)]. Briefly, 10 µg of protein was mixed with potassium phosphate buffer (75 mM, pH 7.0) and 10 mM H₂O₂ to a volume of 1 mL. The rate of H₂O₂ disappearance was detected by measuring the absorbance at 240 nm every 30 sec for a total of 2 min. One unit of catalase activity was defined as the amount of catalase required to degrade 1 µmole H₂O₂ per min.