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利用细胞表面生物素化检测原代小鼠肝细胞表面低密度脂蛋白受体(LDLR)的实验方案。

Protocol for detecting LDLR on the cell surface of primary mouse hepatocytes using cell-surface biotinylation.

作者信息

Ma Fanglin, Cai Bishuang

机构信息

Division of Liver Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Division of Liver Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

出版信息

STAR Protoc. 2025 Jul 23;6(3):103977. doi: 10.1016/j.xpro.2025.103977.

Abstract

Here, we present a protocol for selective biotinylation, solubilization, and enrichment of plasma membrane proteins from primary mouse hepatocytes. We describe steps for injecting C57/BL6 mice via the tail vein with adeno-associated virus subtype 8 (AAV8) to specifically knock down the target gene in hepatocytes. We then detail procedures for labeling surface proteins with EZ-LINK Sulfo-NHS-LC-Biotin and enriching low-density lipoprotein receptor (LDLR) with immunoprecipitation. Finally, we outline procedures for assessing surface LDLR with streptavidin-HRP. For complete details on the use and execution of this protocol, please refer to Ma et al..

摘要

在此,我们展示了一种从原代小鼠肝细胞中选择性生物素化、溶解和富集质膜蛋白的方案。我们描述了通过尾静脉向C57/BL6小鼠注射腺相关病毒8型(AAV8)以特异性敲低肝细胞中靶基因的步骤。然后,我们详细说明了用EZ-LINK Sulfo-NHS-LC-生物素标记表面蛋白并用免疫沉淀法富集低密度脂蛋白受体(LDLR)的程序。最后,我们概述了用链霉亲和素-辣根过氧化物酶评估表面LDLR的程序。有关本方案使用和实施的完整详细信息,请参考Ma等人的文献。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b7be/12305577/90b53df8a56a/fx1.jpg

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