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金虫 (小有名气)

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虫号: 472482
注册: 2007-12-04
专业: 天然药物化学

[求助] 梯度离心提取心肌细胞细胞膜

我在文献上得到一个提取心肌细胞细胞膜的的protocol，
1g of ventricular tissue was washed in ice-cold 0.9% NaCl and homogenized in ice-cold buffer containing 0.6 M sucrose and 10 mM imidazole-HCl (pH 7.0). The homogenate was centri-fuged at 1,000 g for 5 min. The supernatant was centrifuged
at 12,000g for 30 min. The 12,000 g supernatant was diluted in 1.5 volumes of 160 mM NaCl and 20 mM HEPES-Tris (pH 7.4). This vesicle suspension was brought up to 30 ml with the same solution supplemented with 0.25 M sucrose. The fraction was then centrifuged at 160,000 g for 70 min. The pellet representing the sarcolemma-enriched fraction
请问，这里1000*g， 12,000*g 都是用来做什么的啊，然后12,000g的上清液加入1.5倍的160 mM NaCl and 20 mM HEPES-Tris (pH 7.4)是干什么呢，为什么又要扩容到30ml呢？这个30 ml很重要吗，我能够根据自己处理的组织体积进行体积的增减吗，毕竟我用的离心管只有10 ml 啊。我其实做过这个实验，30ml 和10ml的扩容体积感觉造成了很大差别，这是为什么啊。生物方面的菜鸟，多谢帮助啊。

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