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wovs211木虫 (正式写手)
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[求助]
有关植物逆境胁迫下ASA-GSH关键代谢酶的测定
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各位虫友，关于ASA-GSH代谢关键酶MDHAR的测定方法，我用的是下面的方法，饭是我测的黑麦草根系中这个MDHAR的OD值没有变化，不知道是怎么回事，十分着急，望虫友们提供帮助。 我的参考文献是Exogenous sodium nitroprusside and glutathione alleviate copper toxicity by reducing copper uptake and oxidative damage in rice (Oryza sativa L.) seedlings，Protoplasma (2014) 251:1373–1386。酶的提取液方法是To extract enzymes, fresh leaf samples (0.5 g) were homogenized separately with a reaction mixture containing 50 mM Kphosphate buffer (pH 7.0), 100 mM KCl, 1 mM AsA, 5 mMβ-mercaptoethanol, and 10 % (w/v) glycerol in pre-chilled mortars and pestles. The homogenate was centrifuged at12,500×g for 15 min and the resultant supernatants werecollected for analysis of enzyme activities and protein content.All procedures were performed at 0–4°C. 我提取的酶液定容到3ml.MDHAR (EC 1.6.5.4) activity was determined according to the method of Hossain et al. (1984). The reaction mixture contained 50 mM Tris–HCl buffer (pH 7.5), 0.2 mM NADPH, 2.5 mM AsA, 1.0 U of AO and enzyme solution in a final volume of 0.7 ml. The activity was calculated from the change in ascorbate at 340 nm for 1 min using the extinction coefficient of 6.2 mM−1 cm−1. 各位虫友，我测出OD值没有变化啊，怎么办啊。望虫友们提供帮组，谢谢。 |
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