| Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons
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Author:
Date:
2018-01-05
[Abstract] Quantitative analysis of proteins secreted from the cells poses a challenge due to their low abundance and the interfering presence of a large amount of bovine serum albumin (BSA) in the cell culture media. We established assays for detection of mutant huntingtin (mHtt) secreted from Neuro2A cell line stably expressing mHtt and rat primary cortical neurons by Western blotting. Our protocol is based on reducing the amounts of BSA in the media while maintaining cell viability and secretory potential, and concentrating the media prior to analysis by means of ultrafiltration.
[摘要] 由细胞分泌的蛋白质的定量分析由于它们的丰度低和在细胞培养基中干扰大量牛血清白蛋白（BSA）的存在而提出挑战。 我们建立检测突变亨廷顿蛋白（mHtt）检测分泌Neuro2A细胞系稳定表达mHtt和大鼠原代皮层神经元蛋白质印迹。 我们的方案是基于降低培养基中BSA的量，同时保持细胞活力和分泌潜能，并在通过超滤分析之前浓缩培养基。
【背景】许多蛋白质通过各种分泌途径从细胞分泌到细胞外环境中。这些途径包括在ER-高尔基体 - 质膜途径之后的常规分泌途径（Lee等，2004）和多个非常规途径，例如溶酶体胞吐作用，穿过质膜的易位和外泌体以及胞外体释放（Zhang和Schekman，2013）。为了研究这些途径，经常需要分析培养细胞分泌的蛋白质进入培养基。蛋白质可以游离形式分泌，也可以与胞膜结构如胞外体和外泌体结合（Zhang and ...
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| Zonal Sedimentation Analysis on Sucrose Gradients
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Author:
Date:
2014-04-20
[Abstract] Zonal sedimentation analysis on sucrose gradients allows estimation of the molecular size of an individual protein or a protein complex by centrifugation at a constant speed under nondenaturing conditions. This method is particularly suitable for globular proteins like the influenza A virus (IAV) protein hemagglutinin (HA). Here, I describe step by step a protocol used to evaluate the oligomeric state of recombinant HA trimers (Magadan et al., 2013).
[摘要] 对蔗糖梯度的区域沉降分析允许通过在非变性条件下以恒定速度离心来估计单个蛋白质或蛋白质复合物的分子大小。 该方法特别适用于如甲型流感病毒（IAV）蛋白血凝素（HA）的球状蛋白。 在这里，我逐步描述用于评估重组HA三聚体的寡聚状态的方案（Magadan等人，2013）。
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| Antibody Purification from Western Blotting
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Author:
Date:
2012-03-20
[Abstract] This protocol describes a method of purifying antibodies from sera with denatured antigens immobilized on western blot membranes. Advantages include (1) fast and easy; (2) purification of antibody with antigen in denatured form allows high yield in case antigen protein solubility is limited. Disadvantage is that possible antibodies that recognize certain 3D structure in solution of antigens might not be purified using such a method. Regarding this issue, the flow through is recommended to be kept and can be used for other purification methods with folded antigens.
[摘要] 本指南介绍了一种从固定在免疫印迹膜上含有变性抗原的血清中纯化抗体的方法。优点包括：(1)快速且容易；(2)抗原蛋白溶解度有限的情况下从含变性抗原的血清中纯化抗体产量更高。缺点是抗体识别抗原的可能性三维结构在抗原溶液中无法用这种方法纯化出来。由于这个原因，建议保留过滤液，可以用其他纯化方法纯化出折叠抗原。
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