| Morphological Quantification of Nuclei and Mitochondria in Serial Block-face Scanning Electron Microscopy Images
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Author:
Date:
2015-09-20
[Abstract] Serial Block-face Scanning Electron Microscopy (SBF-SEM or 3D-EM) is a powerful tool to study biological structure in ultrastructural level. Quantification of cellular ultrastructure is useful to providing biological information. This technique requires not only high quality of tissue fixation and ideal sample embedding to preserve structures, but also delicate 3D image scanning and post-processing of images. We have adapted previous method to optimize the EM technique to detect and study cellular ultrastructure. Here we present the method to embed samples for 3D-EM technique and to quantify the morphological parameters of nucleus and mitochondria.
Part I. Tissue embedding for 3D-EM images
[摘要] 串行块面扫描电子显微镜（SBF-SEM或3D-EM）是研究超微结构水平生物结构的强大工具。 细胞超微结构的定量对于提供生物信息是有用的。 这种技术不仅需要高质量的组织固定和理想的样品嵌入以保存结构，而且还需要精细的3D图像扫描和图像的后处理。 我们改编以前的方法优化EM技术检测和研究细胞超微结构。 在这里我们提出嵌入样品的3D-EM技术和量化的核和线粒体的形态参数的方法。
第I部分：3D-EM图像的组织嵌入
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| In vitro Detection of S-acylation on Recombinant Proteins via the Biotin-Switch Technique
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Author:
Date:
2014-11-20
[Abstract] Protein palmitoylation is the post-translational modification of proteins via the attachment of palmitate through acyl linkages. The nucleophile sulfhydryl group of cysteines is the common palmitoylation site. Covalent attachment of palmitate occurs on numerous proteins and is usually associated with directing protein localization to the endomembrane system. Detection of protein palmitoylation by in vivo labeling with tritium-labeled palmitic acid typically requires an autoradiographic exposure time of several months, and, thus is not suitable for rapid analyses. Here, we described an easy protocol for quick in vitro detection of protein S-acylation using the Arabidopsis protein kinase, PBS1, as an example. To determine whether PBS1 is modified through thioester ...
[摘要] 蛋白质棕榈酰化是通过棕榈酸酯通过酰基键连接的蛋白质的翻译后修饰。半胱氨酸的亲核巯基是常见的棕榈酰化位点。棕榈酸酯的共价附着发生在许多蛋白质上，并且通常与将蛋白质定位到内膜系统相关。通过体内标记氚标记的棕榈酸来检测蛋白质棕榈酰化通常需要几个月的放射自显影曝光时间，因此不适合快速分析。在这里，我们描述了使用拟南芥蛋白激酶（PBS1）作为实例的快速体外检测蛋白S-酰化的简单方案。为了确定PBS1是否通过硫酯键连接到酰基修饰，我们采用"生物素开关"测定法（Hemsley等人，2008）。这项工作首次发表在Qi。et al。（2014），但我们在这里扩展方法。 PBS1在植物的基础免疫系统内起作用，并且是细菌半胱氨酸蛋白酶AvrPphB的靶（Shao等人，2002; Zhang等人，2010） 。它含有预测的N-末端S - 酰基化基序（MGCFSCFDS），其中Cys-3和Cys-6残基预测为被CSS-Palm 3.0棕榈酰化（http：//csspalm.biocuckoo。 org /; Ren等人，2008）。我们的方法利用羟胺诱导的硫酯键裂解，这导致游离的巯基，然后可以与生物素衍生物1-生物素酰氨基-4- [4' - （马来酰亚胺甲基）环己烷甲酰胺基] - 丁烷（生物素-BMCC）缀合。通过蛋白质印迹用链霉亲和素 - ...
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