蛋白质组学

核蛋白提取

 

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

咋没有人理我呢

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 MgCl2
25mM KCl
10mM HEPES(pH 7.5)
protease inhibitors 1:200
Buffer B: 0.25mM sucrose in Buffer A
Buffer N: 1.1mM sucrose in Buffer A
PBS:137mM NaCl
2.7mM KCl
2mM KH2PO4
10mM Na2HPO4
pH=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10min
4、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10min
7、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

这是我的protocol,包括胞浆,胞核,线粒体蛋白挺好用的。

Preparation of mitochondrial extracts
Mitochondria 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 l 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 l 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l 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℃.

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 g
KcL(74.55) 10mM 0.07455
MgCl2(203.3) 1.5mM 0.030495
EDTA-Na2(372.24) 1mM 0.037224
DTT(154.25) 1mM 0.015425
DMSF(174.2) 1mM 0.01742
pH 7.5 100ml
核裂解液的配制
Hepes(238.3) 20mM 0.09532 (g)
Glycerol(甘油) 25% 5ml
NaCl(58.44) 420mM 0.490896
MgCl2(203.3) 1.5mM 0.006099
EDTA-Na2(372.24) 0.2mM 0.00148896
PMSF(174.2) 0.5mM 0.001742
DTT(154.25) 0.5mM 0.0015425
Leupetia 5 ug/ml

20 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。

多谢各位

谢谢侬哦

请问在组织中怎么提取核蛋白呢?

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