| In vitro Microtubule Binding Assay and Dissociation Constant Estimation
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Author:
Date:
2016-03-20
[Abstract] Microtubules (MTs) support an astonishing set of versatile cellular functions ranging from cell division, vesicle transport, and cell and tissue morphogenesis in various organisms. This versatility is in large mediated by MT-associated proteins (MAPs). The neuronal MAP Tau, for example, is stabilizing MTs in axons of the vertebrate nervous system and thus provides the basis for enduring axonal transport and the long life span of neurons (Mandelkow et al., 1994). Tau has been shown to bind to MTs directly in vitro and also to promote their nucleation from α-/β-tubulin subunits (Goode et al., 1994). Recently, we identified a plant-specific protein family called “companion of cellulose synthase” (CC), which was shown to bind MTs and enhance dynamics of the cortical ...
[摘要] 微管（MT）支持惊人的一套多功能细胞功能，从细胞分裂，囊泡运输，以及细胞和组织形态发生在各种生物体。这种多功能性大量由MT相关蛋白（MAP）介导。例如，神经元MAP Tau是稳定脊椎动物神经系统轴突中的MT，因此为持久的轴突运输和神经元的长寿命提供了基础（Mandelkow等人，1994）。 Tau已经显示在体外直接结合MT，并且促进它们从α-/β-微管蛋白亚基的成核（Goode等人，1994）。最近，我们鉴定了称为"纤维素合酶的伴侣"（CC）的植物特异性蛋白质家族，其被证明结合MT并在盐胁迫下增强植物细胞中皮质MT阵列的动力学（Endler等，2015）。因此，CC被假定为帮助植物细胞应对胁迫条件，从而在不利生长条件下维持生物量生产。在这里，我们提供详细的实验信息在体外 MT结合测定，其允许评估感兴趣的蛋白质是否绑定到MT。该测定法在自旋向下方法中利用高分子量的MT，并且使得能够测定解离常数K d sub，其是蛋白质与MT的结合强度的量度。
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| Detection of Hog1 Phosphorylation in Candida albicans in Response to an Antifungal Protein
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Author:
Date:
2014-09-20
[Abstract] It is becoming increasingly apparent that stress signalling is important for tolerance of fungal species to antifungal chemicals and proteins. The high-osmolarity glycerol (HOG) pathway responds to a number of stressors including osmotic and oxidative stress. This protocol describes a method to detect activation of the Candida albicans (C. albicans) MAPK Hog1 by monitoring its phosphorylation in response to an antifungal protein.
[摘要] 越来越明显的是，应激信号传导对于真菌物种对抗真菌化学品和蛋白质的耐受性是重要的。 高渗透压甘油（HOG）途径对包括渗透和氧化应激在内的许多应激物起反应。 该方案描述了通过监测其对抗真菌蛋白的磷酸化来检测白色假丝酵母（embiculans）（白色念珠菌）MAPK Hog1的活化的方法。
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| Enzymatic Reactions and Detection of C3 Cleavage Fragments
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Author:
Date:
2014-08-20
[Abstract] The complement component C3 is the major effector molecule of the complement system. C3 circulates in the blood and interstitial fluids as pro-enzyme and is activated by enzymatic cleavage into a C3a portion, a classic anaphylatoxin that functions as chemoattractant and immune cell activator, and the C3b portion, the body’s most potent opsonin. C3 cleavage is in most cases mediated by an enzyme complex called the C3 convertase. However, it is now becoming increasingly clear that the cleavage of C3 by a range of ‘single’ proteases into bioactive C3a and C3b fragments is of high physiological significance. Here, we describe a protocol for the enzymatic cleavage of human C3 by the serine protease cathepsin L and the detection of the cleavage products C3a and C3b by western blotting as an ...
[摘要] 补体组分C3是补体系统的主要效应分子。 C3作为前酶在血液和间质液中循环，并通过酶裂解活化为C3a部分，用作化学引诱物和免疫细胞活化剂的经典过敏毒素和作为机体最有效调理素的C3b部分。 C3切割在大多数情况下由称为C3转化酶的酶复合物介导。 然而，现在越来越清楚的是，通过一系列"单一"蛋白酶将C3切割成生物活性C3a和C3b片段具有高生理学意义。 在这里，我们描述了通过丝氨酸蛋白酶组织蛋白酶L酶切割人C3的方案和通过蛋白质印迹检测裂解产物C3a和C3b作为这种酶反应的实例。
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