| CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae
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Author:
Date:
2017-05-20
[Abstract] Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting efficiency of PCS technology is not high enough to achieve multiple splitting at a time. This protocol describes a procedure for achieving simultaneous and multiple chromosome splits in the budding yeast Saccharomyces cerevisiae by a new technology called CRISPR-PCS. At least four ...
[摘要] 染色体工程是应用于基础生物学和生物技术的重要技术。染色体分裂技术称为PCS（PCR介导的染色体分裂）已经被开发为发芽酵母中的基本染色体工程技术。然而，PCS技术的分割效率不够高，不能一次实现多次分割。该协议描述了通过称为CRISPR-PCS的新技术在芽状酵母酿酒酵母中实现同时和多个染色体分裂的过程。基因组中至少四个独立的位点可以通过一次转化来分裂。与常规PCS技术相比，获得多重分裂酵母菌株的总时间和劳动力大大降低。
背景 能够快速有效地操纵多个遗传基因座或染色体区域的染色体工程技术变得越来越重要。这些技术为阐明染色体和基因组功能提供了有力的手段。此外，它可以用于通过创建广泛的遗传变体来繁殖有用的菌株。在芽殖酵母酿酒酵母（Saccharomyces ...
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| PNGase Sensitivity Assay to Study the Folding Status of Proteins
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Author:
Date:
2016-10-05
[Abstract] This protocol aims to evaluate folding status of proteins, utilizing peptide:N-glycanase (PNGase) sensitivity. In the cytosol, PNGase works as a deglycosylation-enzyme. N-glycans on unfolded/misfolded proteins are more susceptible to PNGase than N-glycans on folded proteins because of the preference of PNGase to non-native proteins. PNGase is endogenously expressed in various cell types, including HCT116 cells, DT40 cells and mouse embryonic fibroblast cells. Partial deglycosylation by PNGase can be detected by faster migration of band in SDS-PAGE. You can compare tightness of the folding ...
[摘要] 该协议旨在评估蛋白质的折叠状态，利用肽：N-聚糖酶（PNGase）灵敏度。 在细胞质中，PNGase作为去糖基化酶。 由于PNG酶对非天然蛋白的偏好，解折叠/错折叠蛋白上的N-聚糖比折叠蛋白上的N-聚糖更易受PNG酶的影响。 PNGase在多种细胞类型中内源表达，包括HCT116细胞，DT40细胞和小鼠胚胎成纤维细胞。 通过PNGase的部分去糖基化可以通过在SDS-PAGE中更快的条带迁移来检测。 您可以比较感兴趣的野生型和突变蛋白之间折叠的紧密度。 该方法可以与常规的分子和细胞生物学设备一起使用，但仅应用于糖蛋白。
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| Trypsin Sensitivity Assay to Study the Folding Status of Proteins
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Author:
Date:
2016-10-05
[Abstract] This protocol aims to evaluate folding status of proteins, utilizing trypsin sensitivity. Unfolded/misfolded proteins are more susceptible to trypsin than folded proteins, because trypsin easily accesses and cleaves loosely folded parts of proteins. This method is especially useful to compare tightness of the folding among wild-type and mutant proteins. As trypsin generally cleaves a peptide bond at the carboxyl-terminal side of the amino acids lysine or arginine, this method can be used to analyze the folding status of different types of proteins such as integral membrane or soluble proteins ...
[摘要] 该协议旨在评估蛋白质的折叠状态，利用胰蛋白酶敏感性。 由于胰蛋白酶容易进入和切割松散折叠的蛋白质部分，展开的/错误折叠的蛋白质比折叠的蛋白质更易于胰蛋白酶。 这种方法特别适用于比较野生型和突变型蛋白质之间折叠的紧密度。 由于胰蛋白酶通常在氨基酸赖氨酸或精氨酸的羧基末端侧切割肽键，所以该方法可用于分析不同类型蛋白质如整合膜或可溶性蛋白质的折叠状态（Ninagawa等，2015 ），适用于任何物种和组织以及重组蛋白的细胞裂解物。 您可以使用这种技术与常规分子和细胞生物学设备。
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