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Potassium Glutamate (C5H8KNO4·H2O)

L-谷氨酸钾盐一水合物

Company: Sigma-Aldrich
Catalog#: G1501
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Self-organization Assay for Min Proteins of Escherichia coli in Micro-droplets Covered with Lipids
Author:
Date:
2020-03-20
[Abstract]  The Min system determines the cell division plane of bacteria. As a cue of spatiotemporal regulation, the Min system uses wave propagation of MinD protein (Min wave). Therefore, the reconstitution of the Min wave in cell-sized closed space will lead to the creation of artificial cells capable of cell division. The Min waves emerge via coupling between the reactions among MinD, MinE, and ATP and the differences in diffusion rate on the cell membrane and in the cytoplasm. Because Min waves appear only under the balanced condition of the reaction-diffusion coupling, special attentions are needed towards several technical points for the reconstitution of Min waves in artificial cells. This protocol describes a technical method for stably generating Min waves in artificial cells. [摘要]  [摘要 ] Min系统确定细菌的细胞分裂平面。作为时空调节的提示,Min系统使用MinD 蛋白的波传播(Min wave)。因此,Min波在细胞大小的封闭空间中的重构将导致能够分裂细胞的人造细胞的产生。闵波出现经由耦合之间反应小号中MinD的,的MinE ,和ATP 和所述differenc ES 在细胞膜上的扩散速度和在细胞质中。因为最小波仅在反应扩散耦合的平衡条件下出现, 特别关注,需要对几个技术要点为闽波在人造细胞重建。该协议描述了一种在人造细胞中稳定产生Min波的技术方法。

[背景 ] 敏系统,它决定了细胞分ER 对称细胞分裂,是在细菌细胞内的组织系统的最显着的例子之一(Rothfield 等人,2005;和罗利特马戈林,2013年)。敏系统使用图案形成在细胞内的时间依赖性蛋白梯度的公知的作为敏波(宽松等人,2008; Halatek和Frey,2012;邦尼等人,2013; Zieske 。等人,2016 ; Kohyama 。等人, 2019 )。Min波是由两种蛋白MinD 和MinE 的反应扩散耦合产生的。通过与ATP结合,MinD 形成二聚体并附着在膜上。的MinE 被招募到的ATP MinD的和诱导ATP酶的活性MinD的。通过MinE ,ATP- MinD 变为ADP- MinD ,并从膜上脱离。ADP- MinD的被转换回ATP- ...

Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX)
Author:
Date:
2016-08-20
[Abstract]  The lipid Phosphatidylinositol-3-phosphate [PtdIns3P or PI(3)P] plays many membrane trafficking roles and is primarily produced by the Class III PI3K, VPS34. Determining the level of cellular PI(3)P however can be complex. Extraction of cellular lipids by methanol/chloroform can struggle to separate and identify distinct phospholipid species. Alternately mass spectrometry may be utilised but this requires significant set up of specialised equipment and time to utilise.

Use of a PI(3)P-binding-specific recombinant protein domain is a quick method for ascertaining cellular PI(3)P levels and can also allow visualisation of sub-cellular localisation. The PX domain of p40phox (herein referred to as PX) is very specific for PI(3)P over other phospholipid species (Kanai et al., ...
[摘要]  脂质磷脂酰肌醇-3-磷酸[PtdIns3P或PI(3)P]扮演许多膜贩运角色,主要由III类PI3K,VPS34产生。然而,确定细胞PI(3)P的水平可以是复杂的。通过甲醇/氯仿提取细胞脂质可能难以分离和鉴定不同的磷脂种类。或者可以使用质谱法,但这需要大量设置专门的设备和时间来利用。
 使用PI(3)P结合特异性重组蛋白结构域是用于确定细胞PI(3)P水平的快速方法,并且还可以允许亚细胞定位的可视化。 p40phox的PX结构域(本文称为PX)对PI(3)P比其它磷脂种类非常特异(Kanai等人,2001)。然而,在细胞中直接表达PX可能是有问题的,因为它将以显性负性方式作用,以比内源蛋白更大的亲和力结合和螯合PI(3)P,从而干扰细胞途径和PI(3)P水平。因此,使用荧光标记的PX后细胞固定更合适,因为它能突出显示PI(3)富集结构,没有扰乱系统的风险。

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