普利萊 細(xì)胞核-胞漿蛋白-胞膜制備試劑盒 貨號(hào): P1201-50

描述：本試劑盒能夠從哺乳動(dòng)物新鮮或凍存的組織塊、貼壁或懸浮細(xì)胞中制備細(xì)胞核、細(xì)胞 膜與胞內(nèi)質(zhì)膜、細(xì)胞漿等三種主要亞細(xì)胞組分。其獨(dú)特的試劑成分與優(yōu)化的制備方案使細(xì)胞核-胞漿-胞膜制備過程簡單易行，無需特殊設(shè)備和超速離心，可在1小時(shí)內(nèi)完成。應(yīng)用本試劑盒制備的胞膜是細(xì)胞 膜和細(xì)胞器膜如線粒體、內(nèi)質(zhì)網(wǎng)及質(zhì)膜的混合物；得到的細(xì)胞核是完整的未裂解細(xì)胞核，胞漿組分為可溶性胞漿蛋白。制備得到的產(chǎn)物純度可勝任后續(xù)的免疫沉淀、蛋白印跡、2-D gel、酶活性檢測和受體分析等實(shí)驗(yàn)。

規(guī)格：50次  100次

儲(chǔ)存： 4℃  12個(gè)月有效

操作步驟：

1、 樣本預(yù)處理

2、  組織細(xì)胞勻漿裂解

3、 粗提物制備

4、 胞膜胞漿制備

5、 細(xì)胞核制備：

說明：

1.        Suspension Buffer 不含去垢劑，重懸于Suspension Buffer 中的胞膜或胞核組分呈不溶解狀態(tài)是正常的，用戶可用自備溶液重懸胞膜或胞核成分。如果進(jìn)行蛋白印跡、2-D get等實(shí)驗(yàn)，用戶可用SDS上樣緩沖直接裂解細(xì)胞 膜或細(xì)胞核成分。對于進(jìn)行免疫共沉淀實(shí)驗(yàn)來說，可用RIPA裂解液（C1053）重懸胞膜或胞核組分。

2.        胞漿組分可直接進(jìn)行蛋白定量。胞核組分如需定量可加入TritonX-100至終濃度1%或SDS至終濃度0.5%用BCA法蛋白定量。

3.        用SDS-PAGE Loading 緩沖液裂解細(xì)胞核后，DNA釋放會(huì)使核裂解物十分粘稠，可95^oC加熱5分鐘，高速震蕩打斷DNA，重復(fù)加熱2-3次。

4.        如果進(jìn)行凝膠阻滯（EMSA）實(shí)驗(yàn)，建議使用普利萊P1200產(chǎn)品，該試劑盒可直接提取可溶性核 蛋白.

5.        試劑盒各組分中未添加蛋白酶抑制劑，用戶可自行選擇添加，通常4^oC快速操作不加蛋白酶抑制劑不會(huì)出現(xiàn)問題。

6.        沉淀胞漿蛋白方法：估算胞漿蛋白組分的體積，加入1/3體積30%三氯醋酸水溶液，-20 ^oC沉淀1小時(shí)或過夜。5,000g, 4 ^oC離心10分鐘，棄上清，空氣略干沉淀。用1xSDS上樣緩沖液溶解沉淀，95^oC加熱5分鐘，上樣電泳。

以下僅列舉數(shù)篇使用普利萊P1201試劑盒發(fā)表的文章供參考。

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15、       Liu X D, Yang J J, Fang D, et al. Functional Upregulation of Nav1. 8 Sodium Channels on the Membrane of Dorsal Root Ganglia Neurons Contributes to the Development of Cancer-Induced Bone Pain[J]. PloS one, 2014, 9(12): e114623.

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