| Choline Uptake Assay in Bacterial Cells
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Author:
Date:
2013-09-20
[Abstract] Choline is a methylated nitrogen compound that is widespread in nature. It is a precursor of several metabolites that perform numerous biological functions and it is predominantly used for the synthesis of essential lipid components of the cell membranes. Since there is no evidence that prokariotes can synthesize choline de novo and because choline uptake from exogenous sources is energetically more favorable than de novo synthesis, bacteria have evolved different uptake mechanisms for choline transport across the bacterial membrane. This protocol describes an easy and high sensitive method to assess choline uptake in bacteria using as tracer [3H]-choline chloride. The protocol was originally intended for Brucella abortus but could be applied for any ...
[摘要] 胆碱是一种在自然界广泛存在的甲基化氮化合物。它是几种代谢物的前体，其执行许多生物功能，并且其主要用于合成细胞膜的必需脂质组分。因为没有证据表明原核生物可以从头合成胆碱，并且因为来自外源的胆碱吸收在能量上比新合成更有利，所以细菌已经对胆碱发展了不同的吸收机制运输通过细菌膜。该方案描述了使用示踪物[3 H] - 氯化胆碱来评估细菌中胆碱吸收的容易和高灵敏度的方法。该方案最初用于
流感布鲁氏菌，但可应用于具有相应修饰的任何细菌，这取决于细菌生长需要（培养基的组成，生长温度， em>）。它可用于确定在不同生长条件下几种细菌物种的胆碱吸收能力。
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| GTP Binding Assays in Arabidopsis thaliana
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Author:
Date:
2013-05-05
[Abstract] Signaling through G proteins constitutes an ancient mechanism that functions in the transduction of extracellular signals into intracellular responses. Activation of a proper receptor by a stimuli leads to an exchange of GDP for GTP, the activation of G proteins and the dissociation of Gα-GTP from the Gβγ dimer for the heterotrimeric G proteins. The G protein subunits remain active until the intrinsic GTPase activity or/and a GTPase activating protein result in the hydrolysis of GTP to GDP and the inactivation of the protein. Here we describe a protocol for measuring GTP binding activity from Arabidopsis plant protein extracts using GTP γ35S. GTP γ35S assay measures the level of G protein activation following a stimuli, by determining the ...
[摘要] 通过G蛋白的信号传导构成了在将细胞外信号转导到细胞内反应中起作用的古老机制。通过刺激激活适当的受体导致GTP的GDP交换，G蛋白的激活和G emα-GTP从G emβγ二聚体的解离异源三聚体G蛋白。 G蛋白亚基保持活性，直到内在的GTP酶活性或/和GTP酶活化蛋白导致GTP水解为GDP和蛋白质的失活。在这里，我们描述了使用GTPγ S，从拟南芥植物蛋白提取物测量GTP结合活性的方案。 GTPγ测定通过测定不可水解的类似物GTPγ35 S的结合活性来测量刺激后G蛋白活化的水平。为了确定特异性G蛋白活性，应包括特异性突变体和/或过表达体提取物，并作为对照测量。
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