Quantifying Lipid Peroxidation and H2O2 in Plant Leaves

Malondialdehyde (MDA) is produced by lipid peroxidation and determined by TBA (thiobarbituric acid) reaction (Hong et al., 2010 (link)). Leaf tissues (0.5 g) were homogenized with 0.1% TCA (W/V) solution in pre-cooled pistil and mortar. The mixture was centrifuged at 12,000 × g for 15 min, and the extract was collected in separate tubes. The reaction solution containing 0.5 ml supernatant, 0.5 ml of 5% TBA and 20% TCA solution incubated in a water bath at 95°C temperature. The reaction stopped by shifting the reaction solution in an ice bath. The absorbance was measured at 532 and 600 nm using spectrophotometer (Hitachi F-4600).
The H₂O₂ contents were observed according to the methodology of Chen et al. (2010) (link). Leaf tissues (0.5 g) homogenized with 5 ml of 0.1% TCA (W/V) solution in pre-cooled pistil and mortar. The mixture was centrifuged and extract was collected in separate tubes. The reaction solution contained 0.5 ml enzyme extract, 0.5 ml of 50-mM PBS and 1 ml of 1 M potassium iodide (KI) solution. The reaction stopped by shifting the reaction solution in an ice bath. The absorbance was measured at 390 nm using spectrophotometer (Hitachi F-4600).