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单胺氧化酶的提纯方法
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求助单胺氧化酶的提纯方法 [ Last edited by 350784478 on 2009-10-10 at 13:33 ] |
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MAO was partially purified by isolation of mitochondria from rat liver homogenates according to Holt et al. (1997). Briefly, male Wistar rats (280–300 g) were euthanised by carbon monoxide (Biomedical Resource Centre, University of KwaZulu-Natal) and livers dissected out, washed in ice-cold potassium phosphate buffer (0.2 M, pH 7.6), and stored at −70 °C until required. Liver tissue (5 g) was homogenized 1:40 (w/v) in 0.3 M sucrose. Following centrifugation at 1000 ×g for 10 min the supernatant was further centrifuged at 10,000 ×g for 30 min to obtain a crude mitochondrial pellet. The pellet was resuspended in 4 ml of 0.3 M sucrose and was layeredonto40mlof1.2Msucrose. A mitochondrial pellet was obtained by centrifugation at 53,000 ×g for 2 h. Fol- lowing a single wash in potassium phosphate buffer; mito- chondria were suspended in 40 ml buffer. |
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