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lisumei666新虫 (小有名气)
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[求助]
大鼠肝微粒怎么制备？？急~~ 已有1人参与
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| 我想制备肝微粒体 ，但是网上流行的版本不一，哪位高人给点指点呀，大鼠的肝微粒体制备过程~~不胜感激！ |
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wendao
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【答案】应助回帖
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qinhy(金币+1): 很好很强大啊~ 2011-08-25 18:17:12
qinhy(金币+1): 很好很强大啊~ 2011-08-25 18:17:12
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方法都差不多，找篇文献你看看，做之前老鼠先灌肝除去血 http://dmd.aspetjournals.org/content/29/3/319.full HLM Preparation. To allow for sufficient microsomes to perform all assays, three livers were prepared for each of the three experiments (the force of the first centrifugation, homogenization buffer, and homogenizing strokes). Approximately 10 g of liver per experimental treatment was allowed to thaw in a room temperature homogenization buffer (0.1 M potassium phosphate buffer, pH 7.4, containing 0.125 M potassium chloride and 1.0 mM EDTA). After transfer to 25 ml of chilled homogenization buffer (plus or minus 0.25 M sucrose in the buffer experiment), livers were minced thoroughly with scissors and homogenized with 10 strokes (6, 8, or 10 strokes in the strokes experiment) using a Teflon-glass homogenizer (870 rpm). Strokes were even and steady, lasting approximately 15 s for passage, except for the first two strokes where greater pressure and time were spent on material on the bottom of the glass tube. The tube was submersed in a small bucket of ice and water during all homogenization. The homogenate was diluted to 4 volumes of sample weight (approximately 40 ml). The samples then were centrifuged at 12,000g (9,000, 10,500, or 12,000gin the force experiment) in a Sorvall RC-5B with a Sorvall SA-600 rotor for 20 min (Sorvall, Newton, CT). The supernatant from the first centrifugation was removed, the mitochondrial pellet was resuspended in 25 ml, and the centrifugation was repeated. The supernatants were combined and centrifuged at 138,000g in a Sorvall Ultra Pro 80 with a Sorvall T-1270 rotor for 60 min. The upper lipid layer was removed and the cytosolic supernatant collected. The microsomal pellet was resuspended in 0.125 M KCl, 0.1 M Tris (pH 7.4) with three homogenization strokes, and the 138,000gcentrifugation for 60 min was repeated. The microsomal pellet was resuspended in incubation buffer with six strokes and brought to a final volume of 26 ml. Samples were stored at −70°C. |
2楼2011-08-22 14:15:14
lisumei666
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3楼2011-08-25 08:55:07
wendao
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lisumei6664楼2011-08-28 21:53:13
lisumei666
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5楼2011-08-29 16:18:36
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