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Tryptone

Company: Sigma-Aldrich
Catalog#: T9410
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Preparation of Bacterial Outer Membrane Vesicles for Characterisation of Periplasmic Proteins in Their Native Environment
Author:
Date:
2020-12-20
[Abstract]  

Bacterial outer membrane vesicles (OMVs) are naturally formed by budding from the outer membrane of Gram-negative bacteria. OMVs consist of a lipid bilayer identical in composition to the original outer membrane and contain periplasmic content within their lumen. Enriched with specific envelope proteins, OMVs make for an excellent native-like platform to study these proteins in-situ using biophysical methods. Here, we describe in detail the preparation of OMVs from Escherichia coli, which are luminally enriched with periplasmic proteins and uniformly labeled with stable isotopes (2H and 15N), suitable for the subsequent characterisation of proteins at atomic resolution in their native environment by solution-state NMR spectroscopy. The ability to perform structural studies of periplasmic

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[摘要]  [摘要]细菌外膜囊泡(OMV)是由革兰氏阴性细菌的外膜出芽自然形成的。OMV由组成与原始外膜相同的脂质双层组成,并且在其内腔中含有周质成分。OMV富含特定的包膜蛋白,是使用生物物理方法原位研究这些蛋白的绝佳天然样平台。在这里,我们详细描述了从大肠杆菌制备OMV的方法,该方法在光亮时富含周质蛋白,并用稳定的同位素(2 H和15 N)均匀标记,适用于后续表征溶液状态NMR光谱分析天然环境中蛋白质的原子分辨率。执行周质成分的结构研究的能力,现场清除的方式来REAC兴的这种独特的细胞室的功能和机理细节的深入了解。

[背景]革兰氏阴性菌的周质是一个相当了不起的细胞室。这个空间中,内和外细菌膜之间禁闭,包含在抽蛋白一个ö ř dinarily高浓度超过300毫克毫升-1 (奥利弗,1996) ,并且在不存在的细胞来源,如ATP,功能几乎大力独立从其胞质对应物。到目前为止,有关周质蛋白的结构知识是使用从其天然环境分离的纯化蛋白专门获得的。因此,这种特殊环境可能对蛋白质施加的任何结构和功能影响在纯化过程中都会丢失。由于周质的体积比低,阻碍了使用生物物理方法如细胞内NMR光谱原位研究周质蛋白的努力,周质的体积比仅占细菌总体积的5-1 0%(Brass等,1986)。 )。

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Quantification of Triphenyl-2H-tetrazoliumchloride Reduction Activity in Bacterial Cells
Author:
Date:
2017-01-20
[Abstract]  This protocol describes the use of the 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) salt to evaluate the cell redox potential of rhizobia cells. The production of brightly colored and insoluble 1,3,5-Triphenyltetrazolium formazan arising from TTC reduction is irreversible and can be easily quantified using a spectrophotometer. This protocol allows the production of reproducible results in a relatively short time for Sinorhizobium meliloti 1021 cells grown both in exponential and stationary phases. The results here presented show that the S. meliloti cells deriving from exponential-phase cultures had increased cell redox potential as compared to the ones deriving from stationary-phase cultures. This means that under exponential growth conditions the S. meliloti ... [摘要]  该方案描述了使用2,3,5-三苯基-2H-四唑氯化物(TTC)盐来评估根瘤细胞的细胞氧化还原电位。由TTC还原产生的鲜色不溶性1,3,5-三苯基四唑甲The的生产是不可逆的,可以使用分光光度计轻松地定量。该方案允许在相对较短的时间内在指数阶段和固定阶段生长的中华根瘤菌中华根瘤菌1021细胞产生可重复的结果。这里提出的结果表明,与来自固定相培养物的细胞相比,从指数阶段培养物得到的猕猴桃细胞具有增加的细胞氧化还原电位。这意味着在指数增长条件下, meliloti 细胞产生更大量的TTC减少所需的还原等同物。

背景 TTC盐是一种水溶性和无色的化合物,可以还原成甲an,高度着色的化合物。甲醛的不可逆形成可以使用分光光度计进行定量。由于其性质和其降低的潜力,该四唑盐广泛用于真核生物和原核生物两者,作为细胞氧化还原活性,活力,药物敏感性和底物利用测定的指标(Byth等人, 2001; Hayashi等人,2003; Raut等人,2008; Lin等人,2008)。由于膜电位,四唑鎓盐的净正电荷有助于细胞摄取,从而允许其细胞内还原(Berridge等人,2005)。在原核生物中,TTC降低的主要研究涉及革兰氏阴性呼吸细菌大肠杆菌,而对Rhizobiacea 家族的成员只有少数研究报道。在这个协议中,这个家族最好的遗传特征成员之一, ...

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