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wo的海宝圆圆新虫 (小有名气)
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[求助]
是关于细胞膜的翻译，求帮忙，
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我只能看出个大概，需要高手能给我详细翻一下，谢谢 Cells from a 1-liter culture were harvested in the late exponential growth phase (cell density, 0.4 to 0.5 g of cells dry [weight] per liter), washed with 150 ml of 100 mM potassium phosphate buffer (pH 7.0), and finally suspended in 10 ml of this buffer. The concentrated cell suspension was diluted with 20ml of 100 mM potassium phosphate buffer (pH 7.0) containing 10 mM MgSO4 and 150 mg of egg lysozyme (E. Merck AG,Darmstadt, Germany). The suspension was incubated for 30 min at 30°C. Subsequently, saturated K2SO4 was added to a final concentration of 0.15 M, which resulted in lysis of the cells. Immediately thereafter, the lysed cell suspension was diluted with 70 ml of 100 mM potassium phosphate buffer (pH 7.0) containing 50 ug of RNase (Miles Laboratories, Ltd., Slough, United Kingdom) per ml (final concentration) and 50 ,ug of DNase (Miles Laboratories, Ltd.), per ml (final concentration). This solution was incubated for 20 min at 30°C, K-EDTA (pH 7.0) was added to a final concentration of 15 mM, and incubation was continued for 10 min. After the addition of MgSO4 (final concentration 20 mM), the mixture was centrifuged (30 min, 48,200 xg, 4°C: first high spin). The pellet containing membranes, cells, and cell debris was resuspended in 25 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 10 mM MgSO4. Whole cells and cell debris were removed by centrifugation (70 min, 750 x g,4°C).The supernatant containing membrane vesicles was carefully decanted. Membrane vesicles were collected by centrifugation (30 min, 48,200 x g, 4C;second high spin). The bright-yellow pellet was resuspended in 50 mM potassium phosphate buffer (pH 7.0) containing 10 mM MgSO4 to a concentration of 35 mg of membrane protein per ml. Aliquots of 0.1 ml were rapidly frozen and stored in liquid nitrogen until use. |
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【答案】应助回帖
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RXMCDM: 金币+1, 多谢应助！ 2014-08-24 08:44:53
wo的海宝圆圆: 金币+10, 翻译EPI+1, ★★★★★最佳答案, 谢谢 2014-08-24 10:24:23
RXMCDM: 金币+1, 多谢应助！ 2014-08-24 08:44:53
wo的海宝圆圆: 金币+10, 翻译EPI+1, ★★★★★最佳答案, 谢谢 2014-08-24 10:24:23
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从一升对数生长期后期之培养细胞收集细胞（细胞密度为每升0.4至0.5克干重），用150ml 100 mM的磷酸钾缓冲液（pH 7.0）洗涤，最后悬浮于10ml该缓冲液。 将该浓缩细胞悬液用含有10 mM MgSO4和150 mg卵溶菌酶（默克公司,达姆施塔特, 德国）的200 ml 100 mM磷酸钾缓冲液（pH 7.0）稀释。细胞悬浮液在30°C孵育30分钟，加入饱和K2SO4至终浓度为0.15 M，以裂解细胞。随即用含有每毫升50微克（终浓度）RNA酶（Miles实验室，斯劳，英国）和每毫升50微克（终浓度）DNA酶（Miles实验室，斯劳，英国）的70ml 100 mM磷酸钾缓冲液（pH 7.0）. 溶液于 30°C孵育20分钟后，加入K-EDTA（pH 7.0）至终浓度为15 mM，并继续孵育10分钟。加入MgSO4（终浓度为20 mM）后，混合液离心（30分钟，48，200 x g， 4°C：第一次高速离心）。含有细胞膜、细胞、细胞碎片的沉淀悬浮于25 ml含有10 mM MgSO4的 50 mM磷酸钾缓冲液（pH 7.0）。通过离心（70分钟，750 x g，4°C）去除（为破碎的）全细胞以及细胞碎片。小心倒出（并收集）含有细胞膜囊泡的上清。（上清再次）离心（30 分钟, 48,200 x g, 4°C：第二次高速离心）。将亮黄色沉淀悬浮于含有10 mM MgSO4的50 mM磷酸钾缓冲液（pH 7.0）中，至终浓度为每毫升35 mg膜蛋白。分装成0.1 ml到小份，快速冷冻并保存在液氮中直至使用。 |
2楼2014-08-24 05:48:27