| Purification of RNA Mango Tagged Native RNA-protein Complexes from Cellular Extracts Using TO1-Desthiobiotin Fluorophore Ligand
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Author:
Date:
2018-04-05
[Abstract] A native purification strategy using RNA Mango for RNA based purification of RNA-protein complexes is described. The RNA Mango aptamer is first genetically engineered into the RNA of interest. RNA Mango containing complexes obtained from cleared cellular native extracts are then immobilized onto TO1-Desthiobiotin saturated streptavidin agarose beads. The beads are washed to remove non-specific complexes and then the RNA Mango containing complexes are eluted by the addition of free biotin to the beads. Since the eluted complexes are native and fluorescent, a second purification step such as size exclusion chromatography can easily be added and the purified complexes tracked by monitoring fluorescence. The high purity native complexes resulting from this two-step purification strategy can ...
[摘要] 描述了使用RNA Mango进行RNA-蛋白质复合物的RNA纯化的天然纯化策略。 RNA芒果适体首先被基因工程改造成感兴趣的RNA。 然后将从清除的细胞天然提取物获得的含有RNA复合物的复合物固定在TO1-Desthiobiotin饱和的链霉亲和素琼脂糖珠上。 洗涤珠粒以去除非特异性复合物，然后通过向珠粒中加入游离生物素来洗脱含RNA芒果的复合物。 由于洗脱的复合物是天然的和荧光的，所以可以容易地添加第二纯化步骤如尺寸排阻色谱，并且通过监测荧光追踪纯化的复合物。 通过这种两步纯化策略产生的高纯度天然复合物可以用于进一步的生物化学表征。
【背景】目前的RNA标签受限于诸如差K ，大尺寸，潜在的生物学干扰或缺乏固有荧光的限制（Panchapakesan等人， 2015年）。 RNA芒果很小，可以简单地整合到茎环结构中，特别是GNRA tetraloops中，它具有生物耐受性，并且最重要的是对其噻唑橙基（TO1）配体TO1-Desthiobiotin（TO1-Dtb）具有高亲和力。 ...
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| Spectrophotometric Determination of Glutamine Synthetase Activity in Cultured Cells
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Author:
Date:
2016-10-05
[Abstract] Glutamine synthetase (GS), which catalyzes the conversion of glutamate and ammonia to glutamine, is widely distributed in animal tissues and cell culture lines. The importance of this enzyme is suggested by the fact that glutamine, the product of GS-catalyzed de novo synthesis reaction, is the most abundant free amino acid in blood (Smith and Wilmore, 1990). Glutamine is involved in many biological processes including serving as the nitrogen donor for biosynthesis, as an exchanger for the import of essential amino acids, as a means to detoxifying intracellular ammonia and glutamate, and as a bioenergetics nutrient to fuel the tricarboxylic acid (TCA) cycle (Bott et al., 2015). The method for the assay of GS enzymatic activity relies on its γ-glutamyl transferase reaction by ...
[摘要] 谷氨酰胺合成酶（GS），其催化谷氨酸和氨转化成谷氨酰胺，广泛分布在动物组织和细胞培养系中。该酶的重要性通过谷氨酰胺，GS-催化的从头合成反应的产物，是血液中最丰富的游离氨基酸的事实提示（Smith和Wilmore，1990）。谷氨酰胺参与许多生物过程，包括作为生物合成的氮供体，作为输入必需氨基酸的交换剂，作为解毒细胞内氨和谷氨酸的手段，以及作为生物能量营养物来给三羧酸（TCA）周期（Bott等人，2015）。用于测定GS酶活性的方法依赖于其γ-谷氨酰转移酶反应，通过测量由谷氨酰胺和羟胺合成的γ-谷氨酰羟肟酸酯，以及反应产物与反应物的色谱分离（Deuel等人 。，1978）。 GS谷氨酰转移酶反应的概述可以在图1中找到。通过分光光度测定法在560nm的特定波长下使用酶标仪测量GS活性。该方法简单，并且具有与应用放射性标记的底物的那些方法相当的灵敏度。该修改的方法已经应用于在包括人乳腺上皮MCF10A细胞和鼠前B FL5.12细胞的培养细胞系中测定/测定GS活性，并且可以用于测量其他细胞系中的GS活性。 >
图1 。GS glutamyl ...
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| Micro Neutralization (MN) Assay of Influenza Viruses with Monoclonal Antibodies
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Author:
Ying Wu, MyungSam Cho, David Shore, Manki Song, JungAh Choi, Tao Jiang, Yong-Qiang Deng, Melissa Bourgeois, Lynn Almli, Hua Yang, Li-Mei Chen, Yi Shi, Jianxu Qi, An Li, Kye Sook Yi, MinSeok Chang, Jin Soo Bae, HyunJoo Lee, JiYoung Shin, James Stevens, SeoungSuh Hong, Cheng-Feng Qin, George F. Gao, Shin Jae Chang and Ruben O. Donis,
Date:
2016-06-05
[Abstract] The human monoclonal antibodies generated from single human B cells were tested to characterize their ability to neutralize virus infectivity. The microneutralization assay is a highly sensitive and specific assay for detecting virus-specific neutralizing antibodies to influenza viruses. This protocol is to measure the ability of human monoclonal antibody to neutralize influenza virus by microneutralization assay.
[摘要] 测试从单个人B细胞产生的人单克隆抗体，以表征其中和病毒感染性的能力。 微量中和测定法是用于检测流感病毒的病毒特异性中和抗体的高度灵敏和特异性的测定法。 该方案是测量人单克隆抗体通过微量中和测定中和流感病毒的能力。
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