抗氧化物的测定2.doc

丙二醛MDA的测定（1）0.05-0.1g叶片液氮研磨后，加入1mL 0.1%TCA，摇晃均匀，加入100μL 5mg/mL的丁羟甲苯，在95度水浴中温浴30分钟然后15000g离心10min, 0.55mL的上清液中加入0.55mL TBA（25%的硫代巴比土酸），然后再在95度水浴中温浴30分钟，在冰上冷却，10000g离心5min将上清液在532nm 测定吸光度，通过扣除600nm下吸收值进行矫正，根据MDA标准曲线定其含量 The level of lipid peroxidation in leaf tissues was determined in terms of the peroxidation byproduct MDA in the samples. Briefly, 50 to 100 mg leaf tissuewas shock frozen in liquid nitrogen and ground in 1mL of 0.1% TCA. To the homogenized samples, 0.1 mL butylated hydroxytoluene (5 mg/mL) was added and the homogenates were incubated at 95_C for 30 min, centrifuged at 15,000g for 10 min, and 0.55 mL of the supernatant fraction was mixed with 0.55 mL of TBA solution (25% thiobarbituric acid). The mixture was heated at 95_C for 30 min, chilled on ice, and then centrifuged at 10,000g for 5 min. The absorbance of the supernatant was measured at 532 nm and corrected by subtracting nonspecific absorption at 600 nm. The amount of MDA was calculated according to an MDA standard curve.From: Specific Roles of a- and g-Tocopherol in Abiotic Stress responses of Transgenic Tobacco（2）0.1g组织浸在2mL 0.1%TCA（三氯乙酸）中，匀浆 10400rpm 离心5min, 每200μL上清液加入800μL 20%的TCA（包含5% TBA），混合物在95度温浴30min迅速在冰上冷却，然后混合物在10400rpm 离心15min，上清液在532nm 测定吸光度。扣除600nm下吸收值进行矫正，用消光系数155Mm-1cm-1计算MDA值Approximately 100 mg of tissue was macerated in 2 ml of 0.1% trichloroacetic acid. The homogenate was centrifuged at 10,400 rpm for 5 min. For every 200 μl of the aliquot of the supernatant, 800 μl of 20% TCA containing 0.5% TBA was added. The mixture was heated at 95?C for 30 min and then cooled quickly in an ice bath. The resulting mixture was centrifuged at 10,400 rpm for 15 min and the absorbance of the supernatant was measured at 532 nm. Measurements were corrected for unspecific turbidity by subtracting the absorbance at 600 nm. The concentration of malondialdehyde content was calculated by using the extinction coefficient of 155 mM?1 cm?1.From: Increased α-tocopherol content in soybean seed overexpres