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muyehenmei

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[求助] 测植物蔗糖合成酶和蔗糖磷酸合成酶时的疑惑?

Enzyme activities were determined in leaf extracts, because in our view this method provides reliable data on the enzyme activity ratios in vivo. A leaf sample (1–1.5 g) was fixed with liquid nitrogen and ground in a mortar. An extracting medium (8–10 ml) containing 50 mM Hepes–NaOH (pH 7.5), 5 mM MgCl2, 1 mM EDTA, 2.5 mM dithiothreitol, and 0.5% BSA was gradually added during grinding. The homogenate was filtered through eight layers of cheesecloth and centrifuged at 20000 g for 15 min. The supernatant was dialyzed for 3 h against a tenfold volume of the extracting medium diluted 2.5 times (this medium was free of BSA but contained 10 mM 2-mercaptoethanol). After dialysis, the extract was used as an enzyme preparation.All procedures were performed at 4℃.

在提取蔗糖合成酶和蔗糖磷酸合成酶时,8层纱布过滤作用是什么?要用透析缓冲液透析3h,作用是什么?可不可以省略这两步啊?求大神指点。有更简单的提取和测量方法吗?
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muyehenmei

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2楼2013-12-16 08:20:48
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水煮鱼麻麻

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【答案】应助回帖

这两部是除去杂质和盐的,是有必要的。我们没有做这两部就做不出来
3楼2015-10-08 11:25:47
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