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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Author:
Date:
2014-12-20
[Abstract] A technique of atomic force microscopy (AFM) called PeakForce quantitative nanomechanical mapping (PeakForce QNM) is an efficient tool for the quantitative mechanobiological imaging of fibrillar aggregate, human epidermal cell and woody plant cell wall topography (Sweers et al., 2011; Heu et al., 2012; Ďurkovič et al., 2012; Ďurkovič et al., 2013). Here, we describe a detailed protocol for the measurement of nanomechanical properties of primary xylem cell walls in woody plants, for the determination of reduced Young’s modulus of elasticity (MOE), adhesion, deformation, and energy dissipation (Figure 1). This new technique provides direct control of the maximum loading force and the deformation depth in cell wall samples keeping indentations small, while ...
[摘要] 称为PeakForce定量纳米机械映射(PeakForce QNM)的原子力显微镜(AFM)技术是用于纤维聚集体,人表皮细胞和木本植物细胞壁形貌的定量机械生物学成像的有效工具(Sweers等人, 2011; Heu等人,2012;Ďurkovič等人,2012;Ďurkovič等人,2013年)。在这里,我们描述一个详细的协议,用于测量木本植物中主要木质部细胞壁的纳米机械性能,用于确定降低的杨氏弹性模量(MOE),粘附,变形和能量耗散(图1)。这种新技术提供了对细胞壁样品中的最大负载力和变形深度的直接控制,保持压痕小,同时消除损伤侧向力,以便保持AFM尖端和植物样品。高分辨率和非破坏性成像为木质植物细胞壁的结构生物学提供了新的定量机械的见解。该程序还可适用于具有变化的刚度范围的其他生物样品。
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