The RiboPuromycylation Method (RPM): an Immunofluorescence Technique to Map Translation Sites at the Sub-cellular Level
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Author:
Date:
2018-01-05
[Abstract] While isotopic labeling of amino acids remains the reference method in the field for quantifying translation rate, it does not provide any information on spatial localization of translation sites. The rationale behind developing the ribopuromycylation method (RPM) was primarily to map translation sites at the sub-cellular level while avoiding detection of newly synthesized proteins released from ribosomes. RPM visualizes actively translating ribosomes in cells via standard immunofluorescence microscopy in fixed and permeabilized cells using a puromycin-specific monoclonal antibody to detect puromycylated nascent chains trapped on ribosomes treated with a chain elongation inhibitor.
[摘要] 尽管氨基酸的同位素标记仍然是用于定量翻译速率的领域中的参考方法,但是其不提供关于翻译位点的空间定位的任何信息。 开发ribopuromycylation方法(RPM)的基本原理主要是在亚细胞水平上绘制翻译位点,同时避免检测从核糖体释放的新合成的蛋白质。 RPM通过使用嘌呤霉素特异性单克隆抗体的固定的和透化的细胞中的标准免疫荧光显微镜可视化主动翻译细胞中的核糖体以检测捕获在用链延长抑制剂处理的核糖体上的嘌呤化新生链。
【背景】几十年来,氨基酸的同位素标记被认为是研究蛋白质翻译的金标准。虽然这种方法已被证明是非常准确的评估翻译率,它没有提供翻译核糖体的位置信息。最近,氨基酸类似物使荧光检测新生链(Dieterich等人,2007)。然而,几乎所有检测到的信号都来自核糖体释放的多肽。我们最初的想法是开发一种方法来标记新生链,同时还束缚于翻译核糖体。
嘌呤霉素(PMY)是一种氨基糖苷类抗生素,模拟带电荷的tRNA Tyr并掺入核糖体A位点。因此,PMY通过核糖体催化 - ...
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Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology
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Author:
Date:
2017-07-20
[Abstract] Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. These large proteins are densely substituted with short glycan chains, which have many important functional roles including determining the hydration and viscoelastic properties of the mucus gel that lines and protects the intestinal epithelium. In this protocol, we comprehensively describe the method for extraction of murine mucus and its analysis by agarose gel electrophoresis. Additionally we describe the use of High Iron Diamine-Alcian Blue, Periodic Acid Schiff’s-Alcian Blue and immune–staining methods to identify and differentiate ...
[摘要] 在肠和呼吸上皮中称为杯状细胞的专门分泌细胞负责粘蛋白的分泌。 粘蛋白是大的重糖基化蛋白质,通常具有高于10μM的分子量。 这些大蛋白质被短聚糖链密集取代,其具有许多重要的功能作用,包括测定粘液凝胶的水合和粘弹性质,其保护肠上皮。 在该方案中,我们全面地描述了通过琼脂糖凝胶电泳提取小鼠粘液的方法及其分析。 此外,我们描述了使用高铁二胺 - 阿尔星蓝,周期酸席夫氏 - 阿尔辛蓝和免疫染色方法来鉴别和区分这些粘蛋白糖蛋白上糖基化的不同状态,特别是侧重于硫酸化和唾液酸化。 【背景】一层粘液保护肠上皮,主要由粘蛋白,水,蛋白质和无机盐组成。粘液屏障的粘性和凝胶状特性使其能够物理保护和润滑粘膜,主要由粘蛋白赋予。粘蛋白是大的重糖基化蛋白质,通常具有高于10μM的分子量。然而,粘蛋白主要用O-聚糖糖装饰,其占分子量的80%。不同的位点特异性和粘蛋白特异性糖基化模式影响粘蛋白和粘液凝胶的性质。众所周知,粘蛋白糖基化在感染和疾病中发生改变(Arike等人,2017; ...
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Alcian Blue – Alizarin Red Staining of Mouse Skeleton
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Author:
Date:
2012-04-20
[Abstract] Our lab has used the Alcian blue – Alizarin red staining method with certain modifications to characterize skeleton deformities in mice lacking Pek/Perk, encoding a translational control eIF2alpha kinase.
[摘要] 我们的实验室使用阿尔西蓝 - 茜素红染色方法与某些修改,以表征骨骼畸形缺乏Pek/Perk,编码翻译控制eIF2alpha激酶的小鼠。
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