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Company: Sigma-Aldrich
Catalog#: 471216
Other protocol()

Fluorescent Detection of Intracellular Nitric Oxide in Staphylococcus aureus
[Abstract]  Nitric Oxide (NO) is a highly-reactive radical gas that can modify a variety of cellular targets in both eukaryotes and bacteria. NO is produced endogenously by a wide variety of organisms: For example, as a cell-signaling molecule in mammals and bacteria via nitric oxide synthase (NOS) enzymes, and as a product of denitrification. As such, it is of great benefit to NO researchers to be able to sensitively detect intracellular NO and stable reactive nitrogen species (RNS) derived from NO. To this end, a protocol for fluorescent detection of intracellular NO/RNS in biofilm cultures of the Gram-positive pathogen Staphylococcus aureus has been optimized using the commercially-available cell-permeable fluorescent stain 4-Amino-5-Methylamino-2’,7’-Difluorofluorescein Diacetate (DAF-FM ... [摘要]  一氧化氮(NO)是一种高反应性的自由基气体,其可以修饰真核生物和细菌中的多种细胞靶标。 NO由多种生物体内源性产生:例如,作为哺乳动物和细菌中的细胞信号分子,通过一氧化氮合酶(NOS)酶,以及作为脱氮的产物。因此,NO研究人员能够敏感地检测来自NO的细胞内NO和稳定的活性氮物质(RNS)是非常有益的。为此,已经使用商业上可获得的细胞可渗透的荧光染料4-氨基-5来优化用于荧光检测革兰氏阳性病原体金黄色葡萄球菌的生物膜培养物中的细胞内NO/RNS的方案 - 甲基氨基-2',7'-二氟荧光素二乙酸酯(DAF-FM二乙酸酯)。该化合物扩散到细胞中并且通过酯酶的细胞内裂解释放弱荧光DAF-FM,其与NO或其它特异性RNS反应以变成高度荧光的(Kojima等人,1999)。虽然使用荧光板读数器进行荧光的定量,但设想该方案可适用于S的细胞内NO/RNS成像。 aureus biofilms by confocal microscopy。同样,这种技术可以被优化用于检测其它生长条件(即浮游生物培养物)和/或其它细菌/古细菌中的细胞内NO/RNS。

Aβ Extraction from Murine Brain Homogenates
[Abstract]  This protocol details beta-amyloid (Aβ) extraction from transgenic murine brain homogenates. Specifically, mechanical homogenization of brain tissue and sequential extraction of both soluble and insoluble proteins are detailed. DEA extracts soluble proteins, such as Aβ isoforms and APP. Formic acid enables extraction of insoluble protein aggregates, such as Aβ isoforms associated with plaques. This procedure produces soluble and insoluble extracts that are amenable to analysis of Aβ species via western blotting and/or enzyme-linked immunosorbent assays (ELISAs), and these results help assess amyloidogenic burden in animals. [摘要]  该方案详述了从转基因鼠脑匀浆中提取β-淀粉样蛋白(Aβ)。 具体来说,详细说明脑组织的机械均质化和可溶性和不溶性蛋白质的顺序提取。 DEA提取可溶性蛋白质,如Aβ异构体和APP。 甲酸能够提取不溶性蛋白质聚集体,例如与斑块相关的Aβ同种型。 该方法产生可溶性和不溶性提取物,其适于通过western印迹和/或酶联免疫吸附测定(ELISA)分析Aβ物质,并且这些结果有助于评估动物的淀粉样变性负担。