【求助】核蛋白提取

dldenglin求细胞核蛋白提取的试剂配方，及步骤，最好是亲自做过的方法，万分感谢dldenglin咋没有人理我呢johndxy117来源不明，貌似是某文献作者传授给师兄的，自己试过几次，纯度很高，在此表示感谢~

密度梯度离心法提核蛋白试剂Buffer A: 1.0mM MgCl225mM KCl10mM HEPES(pH 7.5)protease inhibitors 1:200Buffer B: 0.25mM sucrose in Buffer ABuffer N: 1.1mM sucrose in Buffer APBS:137mM NaCl2.7mM KCl2mM KH2PO410mM Na2HPO4pH=7.4步骤（以下是我的实验笔记）细胞量至少要1个60mm培养皿，产量与细胞量有指数增长规律。以下以一个75ml培养瓶为例。1、在培养液中用scraper将细胞刮下，900r/min 离心6min，弃上清2、用2ml Buffer A 重悬细胞，洗涤一次，900r/min 离心6min，弃上清3、重悬于10倍体积Buffer A（约1ml）,冰上放置10min4、将悬液转入玻璃匀浆器，研磨5组，每组转15圈。（每组间隔将磨杵拔出，使管内上下受到 的研磨均匀）此步为关键步骤，用力不要太小，要感受到一定的摩擦力。（第一次做我也心里没底，不知道用多大力，不过还是成功了）5、1000g/min ，4度下离心 10 min,此时上清为胞浆，可留样做后面的鉴定6、重悬于3体积Buffer N（1.2ml),并将其轻轻覆盖于2体积（0.8ml)Buffer B 上。（注意尽量不要扰动Buffer B,可在覆盖时注意不让枪头脱离液面，边打边抽出枪头。）1500g/min,4度离心10min7、离心之后可看见管底和管中部液面分界处各有一沉淀，中部的沉淀即为核。小心吸出液体（可保留少量液体以减少核丢失），用PBS先重悬底部沉淀（PBS体积依据蛋白浓度需要，我们一般用40ul左右），吸出（可扔掉，也可留样）。8、再用PBS重悬中间沉淀，1500g/min 低温离心10min，以洗去杂质，洗两遍。沉淀即为胞核。PBS重悬胞核（我们也先用40ul左右）。以上，请指正

magichunter

这是我的protocol，包括胞浆，胞核，线粒体蛋白挺好用的。Preparation of mitochondrial extractsMitochondria were prepared as described by Abou-Khalil et al. (1985)(1) 5×108 cells were washed once with 10 ml Grinding medium and collected by centrifugation for 5 min at 800g at 4℃.(Grinding medium : sucrose 250 mM, EDTA 2 mM, BSA 1 mg/ml, pH 7.4)(2) The pellet was re-suspended in 1ml of Grinding medium and sonicated.(3) Nuclei were collected by centrifugation for 12 min at 800g at 4℃ and the nuclear proteins were extracted as previously described.(4) The supernatant was immediately centrifuged for 20min at 8,500g at 4℃. The supernatant obtained contains the cytosolic fraction.(5) The pellet was re-suspended with 100 ml of Buffer S, sonicated and then precipitated by centrifugation for 20 min at 10,000 rcf at 4℃.(Buffer S: sucrose 150 mM, KCl 40 mM, Tris/HCl 25 mM, BSA 1 mg/ml, pH 7.4)(6) Protein concentration in nuclear, cytoplasmic, and mitochondrial fractions were determined according to the Bradford method.Preparation of cytoplasmatic and nuclear extract(1) Cells (107) were washed once with PBS and re-suspended in 500 ml of Hypotonic lysis buffer A. (Hypotonic lysis buffer A: 10 mM HEPES, 10 mM KCl, 0.1 mM MgCl2, 0.1 mM EDTA, 0.1mM DTT, 5mM PMSF, pH 7.9)(1)After 10min, nuclei were collected by centrifugation for 10 min at 500 rcf at 4℃ in a microcentrifuge. The supernatant contains the cytoplasmic fraction.(2)Then, the nuclear proteins were extracted with 500 ml of Buffer B.(Buffer B: 10 mM HEPES, 100 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 5 mM PMSF, pH 7.9).(3)After incubating for 20 min at 4℃, samples were centrifuged at 10,000 rcf at 4℃ for 20 min.(4)The nuclear extracts were then quantified for protein levels according to the method of Bradford (1976) and used immediately for Western blot or kept at -80℃.

magichunter

另外，我一个师兄还提供了一份。1 核外裂解液的配制 (胞浆裂解液)Hepes(238.3) 20mM 0.4766 gKcL(74.55) 10mM 0.07455MgCl2(203.3) 1.5mM 0.030495EDTA-Na2(372.24) 1mM 0.037224DTT(154.25) 1mM 0.015425DMSF(174.2) 1mM 0.01742pH 7.5 100ml核裂解液的配制Hepes(238.3) 20mM 0.09532 (g)Glycerol(甘油) 25% 5mlNaCl(58.44) 420mM 0.490896MgCl2(203.3) 1.5mM 0.006099EDTA-Na2(372.24) 0.2mM 0.00148896PMSF(174.2) 0.5mM 0.001742DTT(154.25) 0.5mM 0.0015425Leupetia 5 ug/ml20 ml操作过程这样1 收集细胞，各加100 ul核外裂解液（胞浆裂解液）冰浴裂解1 h，16 000 rpm，20 min，上清为胞浆提取物。2 取沉淀部分加15 ul核裂解液，冰浴15 min，离心12 000，10 min，上清为核提取物。

dldenglin

多谢各位

mysong109

谢谢侬哦

longlong09

请问在组织中怎么提取核蛋白呢？

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