Dual Fluorescence Cytometry Assay to Assess Cellular Protein Levels
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Author:
Date:
2020-04-20
[Abstract] Expression levels of cellular proteins can be affected by various perturbations, such as genetic knockout of interactors, drug treatments or cell stress. To specifically measure the effects on protein levels post-synthesis under different experimental conditions, it is important to compensate for transcriptional and other upstream changes. Here, we provide a protocol for a dual-fluorescence flowcytometry-based assay to determine protein levels. The protein of interest is genetically linked to enhanced GFP (eGFP) followed by a viral 2A self-cleaving peptide sequence and mCherry. As a result, translation of the reporter construct leads to two fluorescent protein products from the same mRNA template, which enables unambiguous protein expression analysis with normalization across samples.
[摘要] [摘要 ] 表达水平的细胞蛋白质可受各种扰动,如基因敲除交互件,药物治疗小号或细胞压力。具体测量对蛋白质水平合成后在不同的实验条件,重要的是要补偿对于转录等上游的变化在这里,我们提供了一个协议为双- 荧光流。流式细胞仪为基础的检测,以确定蛋白质水平目的蛋白是遗传关联增强GFP(EGFP 其次是病毒2A自身切割肽)序列和mCherry结果,报告子构建体的翻译会导致来自同一mRNA模板的两个荧光蛋白产物,这使得能够进行明确的蛋白表达分析,并对整个样品进行归一化处理。
背景 ] 合成和维护的Cellu 拉尔在p- Roteins取决于多进程,从转录调节,处理和降解mRNA中翻译,折叠,本地化,翻译后修饰和蛋白质降解(沃格尔而且马科特,2012) 。具体研究的。在蛋白水平合成后的细胞扰动的影响,这一点很重要,以弥补变异上游步骤蛋白表达在这里,我们提供了一个协议的双- 荧光流术为基础的检测,以确定蛋白质水平处于稳定状态如前所述(Itakura 等人,2016; Chitwood 等人,2018; Ngo 等人,2019)。目的蛋白在基因上与增强的GFP(eGFP )融合,随后是病毒2A自切割肽序列和一个第二种荧光蛋白,mCherry (图1),由于在2A s时核糖体跳过了肽键,融合构建体的翻译产生了两种蛋白质产物,其比例为1:1。ite:与eGFP 和mCherry ...
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Flow Cytometry Measurement of Glucocerebrosidase Activity in Human Monocytes
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Author:
Date:
2020-04-05
[Abstract] Glucocerebrosidase (GCase) is an important enzyme for the metabolism of glycolipids. GCase enzyme deficiency is implicated in human disease and the efficient measurement of GCase activity is important for evaluating the efficacy of therapeutics targeting this enzyme. Existing approaches to measure GCase activity include whole blood mass spectrometry-based assays, where an internal standard is used to measure the accumulation of ceramide following metabolism of the synthetic substrate C12-glucocerebroside, and the utilisation of fluorescent probes that bind active GCase and/or release fluorescent metabolites upon cleavage by GCase. Here, we describe the application of a fluorescence-activated cell sorter-based assay to efficiently quantitate GCase enzyme activity in the monocyte population ...
[摘要]
[摘要 ] 葡萄糖脑苷脂酶(GCase )是糖脂代谢的重要酶。GCase 酶缺乏症与人类疾病有关,GCase 活性的有效测量对于评估靶向该酶的治疗剂的功效至关重要。现有的测量GCase 活性的方法包括基于全血质谱的分析,其中使用内标测量合成底物C12-葡萄糖脑苷代谢后神经酰胺的积累,以及利用结合活性GCase 和// 的荧光探针的利用。或通过GCase 裂解后释放荧光代谢物 。在这里,我们描述了基于荧光激活细胞分选术的测定方法的应用,以有效地定量人类外周血单核细胞单核细胞群中的GCase 酶活性。细胞渗透性的GCase 衬底5-(Pentafluorobenzoylamino )荧光素二-β-D- 吡喃葡萄糖苷(PFB- FDGlu )提供了一个用于测量的GCase 活性,由此酶裂解产生的绿色荧光PFB-F染料,在FL-检测流式细胞仪的1个通道。使用溶酶体GCase 活性抑制剂,conduritol B-环氧,以确保特异性。该协议为测量活的单个细胞中的GCase 活性提供了一种有利的方法。
[背景 ] 葡萄糖脑苷脂酶(GCase ),由 GBA1基因是一种溶酶体水解酶,可将葡糖神经酰胺转化为葡萄糖和神经酰胺。GBA1 ...
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Analysis of the Effect of Sphingomyelinase on Rubella Virus Infectivity in Two Cell Lines
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Author:
Date:
2018-09-05
[Abstract] Rubella is a mildly contagious disease characterized by low-grade fever and a morbilliform rash caused by the rubella virus (RuV). Viruses often use cellular phospholipids for infection. We studied the roles of cellular sphingomyelin in RuV infection. Treatment of cells with sphingomyelinase (SMase) inhibited RuV infection in rabbit kidney-derived RK13 cells and African green monkey (Cercopithecus aethiops) kidney-derived Vero cells. Our data further demonstrated that RuV used cellular sphingomyelin and cholesterol for its binding to cells and membrane fusion at the step of virus entry. Detailed protocols of our assays, which assess the effects of SMase treatment on RuV infectivity in RK13 and Vero cells, are described.
[摘要] 风疹是一种轻度传染性疾病,其特征是低风热和由风疹病毒(RuV)引起的麻疹样皮疹。 病毒通常使用细胞磷脂进行感染。 我们研究了细胞鞘磷脂在RuV感染中的作用。 用鞘磷脂酶(SMase)处理细胞抑制兔肾衍生的RK13细胞和非洲绿猴( Cercopithecus aethiops )肾源性Vero细胞中的RuV感染。 我们的数据进一步证明RuV在病毒进入的步骤中使用细胞鞘磷脂和胆固醇来结合细胞和膜融合。 描述了我们的测定的详细方案,其评估了SMase处理对RK13和Vero细胞中RuV感染性的影响。
【背景】风疹病毒(RuV)是一种正链RNA病毒,属于 Togaviridae 家族中的 Rubivirus 属。该家族有两个属, Rubivirus 和 Alphavirus 。风疹病毒是属的鞋底构件的风疹病毒,而许多病毒,例如塞姆利基森林病毒(SFV)和辛德毕斯病毒(SINV),是归类于甲病毒属。 RuV是风疹和先天性风疹综合征(CRS)的致病因子。风疹的特征是低烧,麻疹样皮疹和淋巴结肿大。它通常是一种轻微的疾病。然而,CRS是一种严重的疾病。 CRS导致在怀孕早期患有风疹的母亲所生的新生儿出现多器官缺陷。白内障,感音神经性听力损失和心血管缺陷在CRS中很常见。
以前的研究表明,细胞膜脂质作为RuV感染的结合或进入因子(Mastromarino ...
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