| Cell Surface Protein Biotinylation and Analysis
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Author:
Date:
2013-08-20
[Abstract] A great way to specifically isolate and quantify proteins in the cell surface membrane is to take advantage of the biotinylation technique. It consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pull-down. Then, the samples are subjected to SDS-PAGE separation, transferred to PVDF membranes and probed with specific antibodies. Quantification of cell surface expression is accomplished by densitometric measurement of the bands corresponding to the protein of interest and subsequent normalization by a membrane protein (as control).
[摘要] 特异性分离和定量细胞表面膜中的蛋白质的好方法是利用生物素化技术。 它包括在裂解细胞之前用生物素试剂标记细胞表面蛋白，并通过NeutrAvidin下拉分离这些标记的蛋白。 然后，将样品进行SDS-PAGE分离，转移至PVDF膜并用特异性抗体探测。 细胞表面表达的定量通过密度测量对应于感兴趣的蛋白质的条带并随后通过膜蛋白质（作为对照）标准化来完成。
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| Sodium Current Measurements in HEK293 Cells
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Author:
Date:
2013-08-20
[Abstract] This protocol is used to measure sodium currents from heterologously transfected cells lines, such as HEK293 cells. Standard whole cell patch clamp technique is used to assess ion channel function. This protocol has been used to study cardiac type Nav1.5 sodium channel, and in particular to compare wild-type and mutant channels related to Brugada Syndrome (BrS). Mutations related to BrS provoke a loss of function of the cardiac sodium channel. This is evidenced by a reduction of sodium current density and/or alterations of the activation or inactivation kinetic parameters, such as a slow recovery from inactivation. These effects could explain the alteration of the cardiac action potential that leads to the characteristic ST elevation observed in the electrocardiogram of the ...
[摘要] 该方案用于测量异源转染的细胞系如HEK293细胞的钠电流。标准全细胞膜片钳技术用于评估离子通道功能。该方案已经用于研究心脏型Na v 1.5钠通道，特别是用于比较与Brugada综合征（BrS）相关的野生型和突变型通道。与BrS相关的突变引起心脏钠通道功能的丧失。这通过钠电流密度的降低和/或活化或失活动力学参数的改变，例如从失活缓慢恢复来证明。这些效应可以解释导致在Brugada综合征患者的心电图中观察到的特征性ST升高的心脏动作电位的改变。该方案具有一些变化，适合于研究与心脏类型钠通道功能的改变相关的其它心律失常以及用于研究其它电压依赖性钠通道。
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