Rice plants from control and Fe stress (300 ppm Fe2+ for 3 weeks) treatments were harvested with liquid nitrogen and stored in -80 °C freezer. Rice shoot tissues were applied in the test.
Grind the rice shoot tissues to fine powder with pre-chilled pestle and mortar in liquid N. Around 50 mg of samples were weighted in 2 ml micro-centrifuge tubes and 1 ml of PBS were added to extract the total antioxidants. After 2 mins of vigorously vortex, the mixtures were placed on ice for 10 mins. Subsequently, the tubes were centrifuged at 14,000 g for 5 mins at 4 °C followed by collecting the supernatants into fresh 1.5 ml micro-centrifuge tubes. To remove the small debris, the tubes were centrifuged again at the same speed for 2 mins.
Load the trolox standards and samples to the micro-plate according to the protocol. Both standards and samples were measured with two replicates. After adding the Cu2+ reagents, the micro-plate was incubated at room temperature in dark for 90 mins. Measure the absorbance of different wells at the wavelength of 570 nm.
The total antioxidant capacity (trolox equivalent) was significantly induced by the excess Fe treatment in rice shoot tissue.
additional notes: ABTRIAL-TVX7B
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Submitted Jan 24 2018