| Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry
|
|
Author:
Date:
2017-01-20
[Abstract] The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway (Figure 1). Usually molecular chaperones exhibit differential affinities with different conformations of the substrate. With the exception of the sHsp family of molecular chaperones, the shift from a high-affinity binding state to the low-affinity ...
[摘要] 稳定其他蛋白质抵抗热聚集的能力是伴侣蛋白质的主要特征之一。分子伴侣与蛋白质的非构象构象结合。通过动态关联和解离循环来触发底物的折叠，这些循环使底物蛋白保持在折叠通路上（图1）。通常分子伴侣表现出与底物不同构象的差异亲和力。除了分子伴侣的sHsp家族之外，从高亲和力结合状态向低亲和力释放状态的转变由ATP结合和水解引发（Haselback和Buchner，2015）。聚集预防测定是一种简单但尚未确定的测定法，用于确定热不稳定蛋白质如麦芽糖糊精葡糖苷酶（MalZ），柠檬酸盐合酶（CS）和Nde I的伴侣活性。这是基于具有伴侣相似活性的蛋白质预防来自热聚集的蛋白质底物（MalZ，CS和Nde I）的前提。在这里，我们描述了使用我们实验室鉴定的两种不同伴侣蛋白，抵抗素和MoxR1的聚合预防测定的详细方案。分析抗性蛋白（hRes）和MoxR1 a结核分枝杆菌蛋白质对预防MalZ /柠檬酸合酶（CS）/ Nde I聚集的影响。
背景 ![]()
图1.分子伴侣的作用机制。柠檬酸合酶通过越来越多的结构化中间体（I <1>，2 ...
|
|
|
| Aβ Extraction from Murine Brain Homogenates
|
|
Author:
Date:
2016-04-20
[Abstract] This protocol details beta-amyloid (Aβ) extraction from transgenic murine brain homogenates. Specifically, mechanical homogenization of brain tissue and sequential extraction of both soluble and insoluble proteins are detailed. DEA extracts soluble proteins, such as Aβ isoforms and APP. Formic acid enables extraction of insoluble protein aggregates, such as Aβ isoforms associated with plaques. This procedure produces soluble and insoluble extracts that are amenable to analysis of Aβ species via western blotting and/or enzyme-linked immunosorbent assays (ELISAs), and these results help assess ...
[摘要] 该方案详述了从转基因鼠脑匀浆中提取β-淀粉样蛋白（Aβ）。 具体来说，详细说明脑组织的机械均质化和可溶性和不溶性蛋白质的顺序提取。 DEA提取可溶性蛋白质，如Aβ异构体和APP。 甲酸能够提取不溶性蛋白质聚集体，例如与斑块相关的Aβ同种型。 该方法产生可溶性和不溶性提取物，其适于通过western印迹和/或酶联免疫吸附测定（ELISA）分析Aβ物质，并且这些结果有助于评估动物的淀粉样变性负担。
|
|
|