| Expression and Purification of the Human Cation-chloride Cotransporter KCC1 from HEK293F Cells for Structural Studies
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Author:
Date:
2021-04-05
[Abstract] Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na+ and/or K+ with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl- by the transmembrane K+ gradient. In humans, these KCCs play vital roles in the physiology of the nervous system and kidney. However, mechanisms underlying the KCCs specific properties remain poorly understood, partly because purification of membrane proteins is challenging. Here, we present the protocol for purifying the full-length KCC1 from HEK293F cells used in our ...
[摘要] [摘要]阳离子-氯化物共转运蛋白（CCC）介导诸如Na +和/或K +的阳离子与氯离子在膜上的耦合，电中性共价。间幼儿中心家庭，K-CL协同转运蛋白（KCC1-KCC4）抽UDE细胞内氯-通过跨膜ķ +梯度。在人类中，这些KCC在神经系统和肾脏的生理中起着至关重要的作用。然而，特定的KCC性质保持基本机制知之甚少，部分是因为膜蛋白的纯化是具有挑战性的。在这里，我们介绍了从我们最近的出版物中使用的HEK293F细胞中纯化全长KCC1的方案（Liu等人，2019）。该程序可适用于功能和结构研究。
[背景]人类溶质载体12（SLC12 ）基因家族编码阳离子的氯化物协同转运蛋白（CCCS）介导Cl组成的电中性同向转运-和阳离子的Na +或（和）K +跨越质膜。根据其转运特性和氨基酸序列定义，CCC可分为几个分支，包括两个Na-K-2Cl协同转运蛋白（NKCC1和NKCC2），一个Na-Cl协同转运蛋白（NCC）和四个K-Cl协同转运蛋白（KCC1-KCC4 ）。CCC在细胞体积调节，肾脏盐分重吸收和神经元GABA能调节中起重要作用。CCC的结构，生化和生物物理研究涉及在去污剂溶解状态下蛋白质生产和稳定方面的挑战。杆状病毒转导HEK293F细胞（BacMam）系统是异源表达由Eric ...
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| Detection of CLEC5A-JEV Interaction by ELISA
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Author:
Date:
2012-10-05
[Abstract] JEV (Japanese encephalitis virus) belonging to Flaiviridae interacts with CLEC5A (C-type lectin domain family 5, member A), a member of C-type lectin associated with DAP12 signaling protein and expressed on myeloid cell, as the same extent as Dengue virus. This protocol is used to perform and determine the interaction between purified JEV particles with human and murine CLEC5A.Fc fusion proteins by ELISA method. The different strain and batch of purified JEV as well as purity of CLEC5A.Fc fusion protein might affect the result of absorbance. Modifications ...
[摘要] 该实验方案的中文版正在准备中．．．
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