Raft-like membranes from the trans-Golgi network and endosomal compartments
Raft-like membranes from the trans-Golgi network and endosomal compartments
复制标题
来自跨高尔基体网络和内体区室的筏状膜
作者：
M. Waugh
本文描述了一种制备富含跨高尔基网络(TGN)和内体隔室的类筏状细胞内膜组分的方法。这项技术的第一步是通过匀浆来破坏细胞，然后通过差异沉积来清除质膜、晚期内小体、线粒体和内质网。随后采用碳酸盐处理、超声波处理和蔗糖密度梯度超速离心法分离目标膜。分离的亚细胞组份含有不到细胞总蛋白的1%，但它高度富含Synaxin-6和Rab11。通常，40-60%的GM1糖鞘脂细胞池和10-20%的细胞总胆固醇与这种浮力膜组分共分。鉴于GM1作为霍乱毒素的细胞表面受体的作用，以及在一些遗传性疾病中TGN-内体间隔中GM1和胆固醇的水平上调，该方法可能被应用于分析富含GM1的细胞膜的疾病相关变化。分离的膜与质膜、TGN和循环内小体的小窝蛋白富集区很好地分开。整个协议可以在短短1天内完成。
This article describes a procedure to prepare a raft-like intracellular membrane fraction enriched for the trans-Golgi network (TGN) and endosomal compartments. The initial step in this technique involves cell disruption by homogenization, followed by clearance of the plasma membrane, late endosomes, mitochondria and the endoplasmic reticulum by differential sedimentation. Carbonate treatment, sonication and sucrose density-gradient ultracentrifugation are subsequently used to isolate the target membranes. The isolated subcellular fraction contains less than 1% of the total cellular proteins, but it is highly enriched for syntaxin-6 and Rab11. Typically, 40–60% of the cellular pool of GM1 glycosphingolipid and 10–20% of the total cellular cholesterol cofractionate with this buoyant membrane fraction. Given the role of GM1 as a cell-surface receptor for the cholera toxin and that levels of both GM1 and cholesterol in the TGN-endosomal compartment are upregulated in some inherited diseases, this protocol can potentially be applied to the analysis of disease-associated changes to GM1-enriched intracellular membranes. The isolated membranes are very well separated from caveolin-rich domains of the plasma membrane, the TGN and recycling endosomes. The entire protocol can be completed in as little as 1 d.
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影响因子：
2.9
作者：
Pike, LJ;Han, XL;Gross, RW
通讯作者：
Gross, RW
影响因子：
3.3
作者：
Kuliawat, R;Kalinina, E;Arvan, P
通讯作者：
Arvan, P
DOI：
10.1016/j.bbalip.2010.03.007
发表时间：
2010-08
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Vetrivel KS;Thinakaran G
通讯作者：
Thinakaran G
DOI：
10.1172/jci15420
发表时间：
2002-06
期刊：
The Journal of clinical investigation
影响因子：
--
作者：
A. Choudhury;M. Dominguez;V. Puri;D. Sharma;K. Narita;C. Wheatley;D. Marks;R. Pagano
通讯作者：
A. Choudhury;M. Dominguez;V. Puri;D. Sharma;K. Narita;C. Wheatley;D. Marks;R. Pagano
影响因子：
11.8
作者：
Chinnapen, Daniel J-F.;Hsieh, Wan-Ting;te Welscher, Yvonne M.;Saslowsky, David E.;Kaoutzani, Lydia;Brandsma, Eelke;D'Auria, Ludovic;Park, Hyejung;Wagner, Jessica S.;Drake, Kimberly R.;Kang, Minchul;Benjamin, Thomas;Ullman, M. David;Costello, Catherine E.;Kenworthy, Anne K.;Baumgart, Tobias;Massol, Ramiro H.;Lencer, Wayne I.
通讯作者：
Lencer, Wayne I.