| 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 among wild-type and mutant proteins of interest. This method can be used with regular molecular and cell biology equipment, but applied only to glycoproteins.
[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 (Ninagawa et al., 2015) and is applicable to cell lysates of any species and tissues as well as to recombinant proteins. You can use this technique with regular molecular and cell biology ...
[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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| ACE-score-based Analysis of Temporal miRNA Targetomes During Human Cytomegalovirus Infection Using AGO-CLIP-seq
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Author:
Date:
2016-04-20
[Abstract] Although temporal regulation of gene expression during the course of infection is known to be critical for determining the outcome of host-virus interactions, systematic temporal analysis of the miRNA targetomes during productive viral infection has been technically challenging due to the large range of miRNA-mRNA cross-talks at the host-virus interface. High-confidence quantifying models of the suppression efficacy in targeting sites by integrating bioinformatics with Argonaute-crosslinking and immunoprecipitation followed by high-throughput sequencing (AGO-CLIP-seq) (Chi et al., 2009) data have been poorly developed. To accurately identify miRNA target sites and calculate the targeting efficacy of miRNA-target interactions, we developed a new bioinformatic quantitation method, ...
[摘要] 尽管已知在感染过程中基因表达的时间调节对于确定宿主 - 病毒相互作用的结果是至关重要的，但是在生产性病毒感染期间对miRNA targetomes的系统时间分析在技术上是具有挑战性的，因为大范围的miRNA- mRNA在主机 - 病毒接口交叉对话。数据通过将生物信息学与Argonaute-交联和免疫沉淀接着高通量测序（AGO-CLIP-seq）数据（Chi等人，2009）数据结合，已经不发达。为了准确地鉴定miRNA靶位点并计算miRNA-靶相互作用的靶向效果，我们开发了新的生物信息学定量方法，即AGO-CLIP-seq富集（ACE） - 评分算法（Kim等， 2015）。在我们的AGO-CLIP-seq分析中包括未感染的对照可以显着提高病毒或人miRNA的真实靶位点识别的准确性，并且在我们的ACE评分方法中提取生产性人巨细胞病毒（HCMV）感染期间的生理学显着变化。因此，我们建议我们新的基于ACE评分的方法可以应用于各种miRNA targetome研究，这将在其他类型的时间背景下进行，如发展阶段，细胞因子或病原体的免疫刺激和其他病毒。
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