| Relative Quantification of NaV1.1 Protein in Mouse Brains Using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) Method
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Author:
Date:
2021-02-05
[Abstract] Densitometric analysis is often used to quantify NaV1.1 protein on immunoblots, although the sensitivity and dilution linearity of the method are usually poor. Here we present a protocol for quantification of NaV1.1 in mouse brain tissues using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) method. MSD-ECL is based on ELISA (enzyme-linked immunosorbent assay) and uses electrochemiluminescence to produce measurable signals. Two different antibodies are used in this assay to capture and detect NaV1.1 respectively in brain tissue lysate. The specificity of the antibodies is confirmed by Scn1a gene knock-out tissue. The calibration curve standards used in this assay were generated with mouse liver lysate spiked with mouse brain lysate, instead of using a recombinant protein. We ...
[摘要] [摘要]尽管该方法的灵敏度和稀释度线性通常较差，但经常使用光密度分析法定量免疫印迹上的Na V 1.1蛋白。这里瓦特E存在的Na进行定量的协议V在使用小鼠脑组织1.1细观量表发现-电化学发光（MSD-ECL）方法。MSD-ECL基于ELISA（酶联免疫吸附测定），并使用电化学发光产生可测量的信号。此测定法中使用了两种不同的抗体来捕获和检测Na V 1.1分别在脑组织中溶解。Scn1a基因敲除组织证实了抗体的特异性。此测定法中使用的校准曲线标准品是用掺有小鼠脑裂解液的小鼠肝裂解液而不是重组蛋白生成的。我们证明该方法是合格的，可用于特异性，准确度和精密度定量的小鼠脑组织中的Na V 1.1 。
[背景]的Na V 1.1，也称为电压门控钠通道的α亚基，I型，是一种跨膜通过对编码蛋白SCN1A基因（Meisler等人，2010 ）。功能性Na V 1.1的表达降低会导致Dravet综合征（DS），这是一种严重的早发性癫痫性脑病（Dravet等，2005）。Na V 1.1在生物样品中的表达已用作DS的非临床药理生物标记物，可以使用免疫印迹的光密度分析进行测量。密度测定法通常不如标准免疫测定法准确和灵敏。此外，由于蛋白质序列的同源性，某些Na V 1.1抗体可能会与其他电压门控钠通道（VGSC）发生交叉反应，包括Na V 1.2，Na V 1.3和Na ...
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| Isolating Liver Mitochondria by Differential Centrifugation
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Author:
Date:
2016-05-20
[Abstract] In addition to methods aimed at the study of mitochondrial function in-situ, a full understanding of mitochondrial function requires their purification from cells or tissues under specific physiological or pathological conditions. This protocol illustrates a sequential procedure to obtain functional mitochondria with high yield from mice liver tissue. Mitochondria obtained with this method can be used to assess different mitochondrial parameters, including oxygen consumption, membrane potential and calcium retention capacity.
[摘要] 除了旨在研究线粒体功能的方法之外，充分了解线粒体功能需要在特定生理或病理条件下从细胞或组织中纯化。 该方案说明从小鼠肝组织获得高产量的功能性线粒体的顺序程序。 用该方法获得的线粒体可用于评估不同线粒体参数，包括氧消耗，膜电位和钙保留能力。
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| Isolating Brain Mitochondria by Differential Centrifugation
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Author:
Date:
2016-05-20
[Abstract] In addition to methods aimed at the study of mitochondrial function in-situ, a full understanding of mitochondrial function requires their purification from cells or tissues under specific physiological or pathological conditions. This protocol illustrates a sequential procedure to obtain functional mitochondria with high yield from mice brain tissue. Mitochondria obtained with this method can be used to assess different mitochondrial parameters, including oxygen consumption, membrane potential and calcium retention capacity.
[摘要] 除了旨在研究线粒体功能的方法之外，充分了解线粒体功能需要在特定生理或病理条件下从细胞或组织中纯化。 该方案说明从小鼠脑组织获得高产量的功能性线粒体的顺序程序。 用该方法获得的线粒体可用于评估不同线粒体参数，包括氧消耗，膜电位和钙保留能力。
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