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Sodium Chloride for analysis, ACS, ISO


Company: AppliChem
Catalog#: 131659
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Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons
[Abstract]  This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of cells, while offering minimal cell death that often occurs during dissociation of differentiated neurons. Cells plated under the appropriate conditions, either in Petri-dishes or in multi-well plates, will develop and establish synaptic contacts over time since the neuronal culture medium provides the nutrients and trophic factors required for differentiation. In this protocol we describe the methodology for the preparation of both cortical and ... [摘要]  该方案旨在标准化海马和脑皮质神经元原代培养物的体外实验。 培养物应从胚胎发育期间分离的细胞制备,当神经元前体细胞尚未完全分化时。 这有助于提高细胞的质量和数量,同时提供通常在分化的神经元解离期间发生的最小的细胞死亡。 在合适的条件下,在培养皿或多孔板中铺板的细胞将随着时间的推移而发展和建立突触接触,因为神经元培养基提供分化所需的营养和营养因子。 在这个协议中,我们描述了制备皮质和海马神经元培养物的方法。
【背景】本方案描述了使用补充有NeuroCultTM SM1的Neurobasal培养基(Chen等人,2008)的大鼠海马和脑皮层神经元的原代培养物的制备。 NeuroCultTM SM1的组成基于B27补充剂的制剂(Brewer等,1993),但是前者的混合物被发现提高了神经元培养的质量,部分地通过用全转运蛋白替代载脂蛋白转运蛋白 Chen et al。,2008)。 此外,NeuroCultTM SM1的化学成分在原始出版物中有更详细的描述,可以更好地控制实验条件。 用化学确定的培养基制备的神经元培养物的特征在于存在低百分比的星形胶质细胞。 通过添加有丝分裂5-氟-2'-脱氧尿苷的化学抑制剂可以防止维持更长时间的培养物中星形胶质细胞的增殖以允许神经元分化。

Extraction and Quantification of Polyphosphate in the Budding Yeast Saccharomyces cerevisiae
[Abstract]  Inorganic polyphosphate (polyP) is a linear polymer present in both prokaryotic and eukaryotic organisms and made from three to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this molecule goes beyond serving as Pi store or energy source to replace ATP. For instance, in yeast polyP levels have been related to stress adaptation and this molecule has been shown to be the substrate for polyphosphorylation of proteins. Here we describe two different methods to purify polyP from the yeast Saccharomyces cerevisiae and the subsequent protocol to quantify polyP levels by spectrophotometrically measuring the Pi generated upon enzymatic hydrolysis of purified polyP. It must be noted that the purification protocol used greatly influences the ... [摘要]  无机多磷酸盐(polyP)是存在于原核和真核生物中的线性聚合物,由通过磷酸酐键连接的三至数百个正磷酸盐残基制成。 这种分子的生物学作用超越了作为P 1储存或能量源以代替ATP。 例如,在酵母中,polyP水平与应激适应相关,并且该分子已经显示为蛋白质多磷酸化的底物。 在这里,我们描述了从酵母酿酒酵母中纯化polyP的两种不同方法和随后的通过分光光度法测量在酶水解纯化的polyP时产生的Pi来定量polyP水平的方案。 必须注意的是,所使用的纯化方案极大地影响所获得的polyP值。

图1. polyP的酶水解

Fluorescence Recovery after Photobleaching (FRAP) Assay to Measure the Dynamics of Fluorescence Tagged Proteins in Endoplasmic Reticulum Membranes of Plant Cells
[Abstract]  In this protocol, we used fluorescence recovery after photobleaching (FRAP) to measure the influence that some mutations and drug treatment have on mobility of a green fluorescent protein (GFP)-fused viral transmembrane protein into endoplasmic reticulum membranes (Serra-Soriano et al., 2014). The proteins of interest were transiently expressed in Nicotiana benthamiana (N. benthamiana) epidermic cells by agro-infiltration. To minimize transient overexpression artifacts, fluorescence intensity values were gathered at 36 hpi using an inverted Zeiss LSM 780 confocal microscope. Only epidermic cells showing moderated expression levels and homogenous distribution through the ER of the GFP-tagged proteins were used for further experiments. To examine the role of actin ... [摘要]  在该协议中,我们使用光漂白(FRAP)后的荧光恢复来测量一些突变和药物治疗对于将绿色荧光蛋白(GFP) - 融合的病毒跨膜蛋白移动到内质网膜中的影响(Serra-Soriano, et al。,2014)。感兴趣的蛋白质通过农杆菌浸润在烟草(Nicotiana benthamiana)(本塞姆氏烟草)表皮细胞中瞬时表达。为了使瞬时过表达伪像最小化,使用倒置Zeiss LSM 780共聚焦显微镜在36hpi收集荧光强度值。只有表现出中等表达水平和通过GFP标记的蛋白的ER的均匀分布的表皮细胞用于进一步的实验。为了检查肌动蛋白聚合在GFP标记的蛋白质的动员中的作用,我们用拉特管素B,肌动蛋白聚合的抑制剂或用DMSO作为对照预处理组织样品。使用所产生的荧光恢复曲线来获得对应于流动级分的最大荧光回收率(MFR)的百分比和最大回收的半衰期(t 1/2)/sub>)值。