Measurement of Extracellular Ca2+ Influx and Intracellular H+ Efflux in Response to Glycerol and PEG6000 Treatments
|
Author:
Date:
2013-09-20
[Abstract] The characteristics of Ca2+ and H+ fluxes may reflect the activities of aquaporins, as the up-regulation of aquaporin activities is directly associated with the decrease in cytoplasmic H+ concentration and increase in cytoplasmic Ca2+ concentration. The higher aquaporin activities can protect cells against osmotic stresses by altering water flow into and out of the cells. In order to confirm the contribution of aquaporins to the cell tolerance to different osmotic stresses, net Ca2+ and H+ fluxes are measured using the noninvasive micro-test technique (NMT). NMT provides the real-time in situ detection of net ion transport across membranes. Here, we describe the protocol of in situ detection of net Ca2+ ...
[摘要] Ca 2+和H sup +通量的特征可反映水通道蛋白的活性,因为水通道蛋白活性的上调与细胞质H的减少直接相关> + 浓度和细胞质Ca 2+浓度的增加。较高的水通道蛋白活性可以通过改变进入和离开细胞的水流来保护细胞免受渗透压力。为了证实水通道蛋白对细胞对不同渗透胁迫的耐受性,使用非侵入性微测试技术(NMT)测量净Ca 2+和H + +通量, )。 NMT提供了跨膜的净离子迁移的实时原位检测。在这里,我们描述了原位检测穿过转化的巴斯德毕赤氏酵母的净Ca 2+和 + 通量的方案。 >细胞响应于甘油和聚乙二醇6000(PEG6000)处理。将转化的酵母细胞加载到在聚-L-赖氨酸溶液(0.1%w/v水溶液)中预处理的盖片上。细胞固定后,微电极位于单层细胞群上方。在由计算机操纵的两个偏移点处测量微伏电压差。使用ASET 2.0和iFluxes 1.0软件,微伏电压差可以转换为离子通量。该方法有望促进NMT在微生物学中的应用。我们非常感谢Younger USA(徐越北京)NMT服务中心对稿件的批判性阅读。
|
|
Construction and Screening of a Transposon Insertion Library of Yersinia enterocolitica (YeO3-R1)
|
Author:
Date:
2012-08-05
[Abstract] The Mu-transposon system is one of the best characterized transposition systems. Under minimal in vitro set-up, Mu transposition requires only a simple reaction buffer, MuA transposase protein, mini-Mu transposon DNA (donor) and target DNA. The reaction proceeds via initial assembly of the transposition complex that directs transposon integration into target DNA with high efficiency and relatively low target site selectivity. These characteristics make the Mu in vitro transposition technology ideal for the generation of comprehensive mutant DNA libraries usable in a variety of molecular biology applications. This technology has successfully been used for DNA sequencing, functional analyses of plasmid DNA and virus genomes, protein engineering for structure/function and ...
[摘要] 串联亲和纯化(TAP)(Pugi等人,2001; Rigaut等人,1999)是使用目标靶蛋白的标记方法的方法两步纯化方案以在天然条件和表达水平下下拉蛋白复合物。 TAP标签由三种组分组成:钙调素结合肽,烟草蚀纹病毒(TEV)蛋白酶切割位点和作为免疫球蛋白G(IgG)结合结构域的蛋白A.该方案从酵母细胞中使用的原始方法(Pugi等人,2001; Rigaut等人,1999)修饰,用于从果蝇头分离蛋白质复合物,以及卵巢表达感兴趣的TAP标记的蛋白质。为了确定果蝇脆弱X蛋白(dFMR1)的体内结合伴侣,我们开发了表达重组形式的具有羧基末端TAP标签的dFMR1的苍蝇的转基因菌株(Tsai和Carstens,2006)。为了确保构建体在野生型水平表达,我们在救援突变不育表型的基因组拯救构建体的上下文中工程化这种形式的标记蛋白质。使用温和条件进行纯化过程以维持天然蛋白质相互作用。对于在果蝇S2细胞培养物中的TAP方法,我们成功地使用了由Tsai和Carstens先前公布的方案(Tsai和Carstens,2006; Bhogal等人,2011)。...
|
|