| Generation of Giant Unilamellar Vesicles (GUVs) Using Polyacrylamide Gels
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Author:
Date:
2020-11-05
[Abstract] Giant unilamellar vesicles (GUVs) are a widely used model system for a range of applications including membrane biophysics, drug delivery, and the study of actin dynamics. While several protocols have been developed for their generation in recent years, the use of these techniques involving charged lipid types and buffers of physiological ionic strength has not been widely adopted. This protocol describes the generation of large numbers of free-floating GUVs, even for charged lipid types and buffers of higher ionic strength, using a simple approach involving soft polyacrylamide (PAA) gels. This method entails glass cover slip functionalization with (3-Aminopropyl)trimethoxysilane (APTES) and glutaraldehyde to allow for covalent bonding of PAA onto the glass surface. After polymerization ...
[摘要] [摘要]巨型单层囊泡（GUV）是一种广泛使用的模型系统，其应用范围包括膜生物物理学，药物递送以及肌动蛋白动力学研究。虽然一些协议已经为他们这一代人在最近几年已开发，利用这些T的echniques涉及带电脂质的类型和生理离子强度缓冲液一直没有得到广泛的广告Ø PTED。Thi的方案描述了使用包括聚丙烯酰胺（PAA）凝胶的简单方法，即使对于带电荷的脂质类型和更高离子强度的缓冲液，也产生了大量的自由浮动GUV。此方法需要使用（3-氨基丙基）三甲氧基硅烷（APTES）和戊二醛对玻璃盖玻片进行功能化，以允许将PAA共价键合到玻璃表面上。PAA聚合后，将凝胶真空干燥。随后，将选择的脂质均匀地分散在干燥的凝胶表面上，并且可以使用具有不同离子强度的缓冲液来重新水化凝胶并形成GUV。该协议对于在生理条件下生产大量由不同脂质组成的自由浮动GUV而言是可靠的。它可以方便地用常用的实验室试剂进行。
[背景】虽然温和的水化和电铸是两个巨的最常用的方法单层囊泡（GUV）的形成，只有少数研究，报告其使用带电脂质类型和斯坦因的生理离子强度缓冲液（等人。，2017; ...
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| Mapping mRNA-18S rRNA Contacts Within Translation Initation Complex by Means of Reverse Transcriptase Termination Sites and RNAseq
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Author:
Date:
2020-08-20
[Abstract] The nucleotides involved in RNA-RNA interaction can be tagged by chemical- or UV-induced crosslinking, and further identified by classical or modern high throughput techniques. The contacts of mRNA with 18S rRNA that occur along the mRNA channel of 40S subunit have been mapped by site-specific UV crosslinking followed by reverse transcriptase termination sites (RTTS) using radioactive or fluorescent oligonucleotides. However, the sensitivity of this technique is restricted to the detection of those fragments that resulted from the most frequent crosslinkings. Here, we combined RTTS with RNAseq to map the mRNA-18S rRNA contacts with a much deeper resolution. Although aimed to detect the interaction of mRNA with the ES6S region of 18S rRNA, this technique can also be applied to map the ...
[摘要] [摘要 ] 可以通过化学或紫外线诱导的交联来标记参与RNA-RNA相互作用的核苷酸，并通过经典或现代的高通量技术对其进行进一步鉴定。沿着40S亚基的mRNA通道发生的18S rRNA与mRNA的接触已通过位点特异性UV交联进行了定位，随后使用放射性或荧光寡核苷酸进行了逆转录酶终止位点（RTTS）。但是，该技术的敏感性仅限于检测由最频繁的交联产生的那些片段。在这里，我们将RTTS与RNAseq结合使用，以更深的分辨率绘制了mRNA-18S rRNA接触图。尽管旨在检测mRNA与18S rRNA的ES6S区域的相互作用，但该技术也可以用于绘制mRNA与其他非编码RNA分子的相互作用（在转录，剪接或RNA介导的转录后调控过程中（例如，snRNA，microRNA和lncRNA）。
[背景 ] 是与非编码RNA的mRNA的相互作用volved mRNA中的生命周期的每个步骤，从它的生物合成和处理进入细胞核至细胞质中的翻译和最终降解。这些相互作用可以在大分子机器核糖体和剪接体，以及在较小的复合物如RISC或者发生（RNA诱导的沉默复合物）或lncRNA介导的基因表达调节期间（皮萨列夫等人，2008; Engreitz 。等人，2014 Sharma ...
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