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Amersham Typhoon 多功能激光成像仪

Company: Cytiva
Catalog#: 29187191
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Site-specific DNA Mapping of Protein Binding Orientation Using Azidophenacyl Bromide (APB)
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Date:
2020-06-20
[Abstract]  The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication fork directs the assembly of the other associated replisome proteins. We have recently determined the orientation of Saccharalobus solfataricus (Sso) Minichromosome maintenance (MCM) helicase at a DNA fork utilizing a site-specific DNA cleavage and mapping assay. Here, we describe a detailed protocol for site-specific DNA footprinting using 4-azidophenacyl bromide (APB). This method provides a straightforward, biochemical method to reveal the DNA binding orientation of SsoMCM helicase and can be applied to other DNA binding ... [摘要]  [摘要 ] 结合在DNA上的DNA结合蛋白的方向决定了复合物中其他相关蛋白的组装,以进行有意的作用。例如,在复制体中,复制叉处的DNA解旋酶的方向指导我们最近通过定点DNA切割和作图分析确定了DNA叉处的Saccharalobus solfataricus (Sso )微型染色体维持(MCM)解旋酶的方向。 使用4-叠氮苯甲酰溴(APB)进行位点特异性DNA足迹的详细协议。此方法提供了一种直接的生化方法来揭示Sso MCM解旋酶的DNA结合方向,并可应用于其他DNA结合蛋白。

[背景 ] DNA复制是其中基因组双链体链分离成两个模板链中,超前和滞后strands.This功能是通过在生命。如同其他环状六聚体的解旋酶结构域的所有的环状六聚体解旋酶的处理,从理论上讲,这些结构域中的任何一个都可以在易位期间朝向复制叉,并且与已知的3'-5 MCM包含两个结构域; N端结构域(NTD)和C端结构域(CTD)。' 易位directionality.T 他MCM解旋酶负载到DNA起源作为具有面向易位期间解旋酶的每个other.The取向管畸形双六聚体确定两个六聚体是否彼此或旁路彼此活性unwinding.Our最近的一篇论文中离解远表明Saccharolobus solfataricus (Sso MCM )通过NTD引导DNA解链(Perera和Trakselis ...

Photoactivable Cholesterol as a Tool to Study Interaction of Influenza Virus Hemagglutinin with Cholesterol
Author:
Date:
2020-02-20
[Abstract]  Non-covalent binding of cholesterol to the transmembrane region of proteins affect their functionalities, but methods to prove such an interaction are rare. We describe our protocol to label the hemagglutinin (HA) of Influenza virus with a cholesterol derivative in living cells or with immunoprecipitated protein. We synthesized a “clickable” photocholesterol compound, which closely mimics authentic cholesterol. It contains a reactive diazirine group that can be activated by UV-illumination to form a covalent bond with amino acids in its vicinity. Incorporation of photocholesterol into HA is then visualized by “clicking” it to a fluorophore, which can be detected in an SDS-gel by fluorescence scanning. This method provides a convenient and practical way to demonstrate cholesterol-binding ... [摘要]  [摘要 ] 胆固醇与蛋白质跨膜区的非共价结合会影响其功能,但很少有证明这种相互作用的方法。我们描述了我们的协议,以活细胞中的胆固醇衍生物或免疫沉淀蛋白标记流感病毒的血凝素(HA)。我们合成了一种“可点击的” 光胆固醇化合物,该化合物紧密模拟真实的胆固醇。它包含一个反应性重氮基团,该基团可以通过紫外线照射激活,并与其附近的氨基酸形成共价键。然后将光胆固醇掺入HA中,通过将其“点击”到荧光团上进行可视化,可以通过荧光扫描在SDS凝胶中检测到。 该方法提供了一种方便实用的方法来证明胆固醇与其他蛋白质的结合,并可能确定结合位点。

[背景 ] 蛋白质与胆固醇的非共价相互作用被认为可以调节许多蛋白质的运输和功能(de Vries 等,2015)。然而,由于这种相互作用的短暂且相当弱的性质,众所周知,胆固醇结合蛋白和各自的结合位点很难鉴定。精细的制作方法,如NMR或晶体需要大量需要被整合到人造脂质膜,并因此只能访问特殊纯化的蛋白质的我捷思实验室。一个简单的程序包括使用商业的“试剂盒”测量纯化的蛋白质中的胆固醇含量(例如,的Amplex 红胆固醇测定试剂盒,分子探针)。但是,这种方法不灵敏,需要用去污剂将蛋白溶解在膜上,去污剂通常会去除非共价结合的脂质。

一个改进是合成的clickable- photocholesterol ...

Protocol for Ribosome Profiling in Bacteria
Author:
Date:
2019-12-20
[Abstract]  Ribosome profiling provides information on the position of ribosomes on mRNA on a genomic scale. Although this information is often used to detect changes in gene expression under different conditions, it also has great potential for yielding insight into the mechanism and regulation of protein synthesis itself. First developed in yeast, ribosome profiling involves the isolation and sequencing of ribosome-protected mRNA fragments generated by nuclease treatment. Since the application of ribosome profiling in bacteria has been problematic, we report here a systematically optimized protocol for E. coli that we have used with success for other bacteria as well. Cells are harvested by flash-freezing cultures directly in liquid nitrogen. After lysis, translation is arrested by high ... [摘要]  核糖体谱分析提供了基因组尺度上核糖体在mRNA上的位置信息。尽管此信息通常用于检测不同条件下基因表达的变化,但它也具有极大的潜力,可深入了解蛋白质合成本身的机制和调控。核糖体谱分析首先在酵母中开发,涉及分离和测序核酸酶处理产生的核糖体保护的mRNA片段。由于核糖体图谱在细菌中的应用存在问题,因此我们在此报告了针对 E的系统优化方案。大肠杆菌,我们也成功地将其用于其他细菌。直接在液氮中通过速冻培养来收获细胞。裂解后,无需使用抗生素即可通过高镁浓度阻止翻译。这些改进消除了通过离心或过滤收集细胞以及使用氯霉素阻止翻译而引起的伪影。这些改进特别适用于核糖体确切位置至关重要的研究,而不仅仅是每个信息的核糖体数量至关重要,例如旨在监测局部伸长率差异的研究。

【背景】根据mRNA固有的特性,例如起始速率,mRNA结构和密码子使用情况,mRNA的翻译效率各不相同。另外,细胞中的生理变化可以改变mRNA翻译的效率。开发了核糖体分析以在蛋白质合成水平上监测基因表达,从而有可能提出有关基因组的哪些区域被翻译,何时被翻译以及如何调节翻译的普遍问题。核糖体图谱最初由Ingolia和Weissman在酵母中开发(Ingolia et ...

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