The Endoplasmic Reticulum (ER) is a sub-cellular membrane network that spans the cytosol
and connects the nuclear membrane to the plasma membrane. Critical cellular functions (such as protein
biosynthesis, oxidation of xenobiotics, and protein transport) occur in the ER. Since ER-derived
proteins account for only a small fraction of the total cellular proteome, and since the majority of
ER-associated proteins are membrane proteins, which are especially difficult to purify, proteomic
analysis of the ER has been a challenging endeavor. To facilitate the study of the ER proteome,
an ER-like fraction of small vesicles, termed microsomes,can be isolated from cell homogenates
by differential centrifugation. However, efficiently extracting proteins from this membrane-rich fraction
is difficult, making a comprehensive proteomic analysis of lipid-rich microsomal samples difficult [1].
Here we describe a method for the efficient extraction of proteins from rat liver microsomes, using
Pressure Cycling Technology (PCT) and the novel chemistry of the Kit. ProteoSolve .
利用壓力循環(huán)技術(shù)(PCT)從動(dòng)物微粒體中提取蛋白
內(nèi)質(zhì)網(wǎng)(ER)是一種亞細(xì)胞膜網(wǎng)絡(luò)結(jié)構(gòu),它位于細(xì)胞質(zhì)并連接著質(zhì)膜和核膜。一些重要的細(xì)胞功能比如蛋白質(zhì)生物合成、異型生物質(zhì)的氧化以及蛋白運(yùn)輸 等均發(fā)生在內(nèi)質(zhì)網(wǎng)(ER)。由于起源于內(nèi)質(zhì)網(wǎng)(ER)的蛋白只占細(xì)胞蛋白總量的很小一部分,而且大多數(shù)與內(nèi)質(zhì)網(wǎng)關(guān)聯(lián)的蛋白均是膜蛋白,因此這些蛋白非常難 以純化,內(nèi)質(zhì)網(wǎng)(ER)的蛋白質(zhì)組學(xué)分析也因而變得非常具有挑戰(zhàn)性。為了方便內(nèi)質(zhì)網(wǎng)(ER)蛋白的研究,一種小囊泡中類(lèi)似于內(nèi)質(zhì)網(wǎng)的片段和微粒體可通過(guò)梯 度離心從細(xì)胞均質(zhì)中分離出來(lái)。但是,試圖從這種膜含量多的片段中有效的提取蛋白是非常困難的,從而對(duì)脂質(zhì)含量多的微粒體樣品進(jìn)行廣泛的蛋白質(zhì)組學(xué)分析也變 得非常困難。本文描述了一種有效的從大鼠肝臟微粒體中提取蛋白的方法,該方法正是利用壓力循環(huán)技術(shù)(PCT)和一種特別的ProteoSolve試劑盒。