| 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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| Cell-based Assays to Monitor AID Activity
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Author:
Date:
2016-02-05
[Abstract] The enzyme Activation induced deaminase (AID) underpins antibody affinity maturation and isotype switching through its mutagenic activity of deaminating deoxycytidine to deoxyuridine in DNA. Subsequent processing of the deoxyuridine initiates the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. Structure-function analysis of AID requires sensitive and biologically relevant methods to measure its various activities. Here we describe simple but effective methods to measure 1) the ability of AID to mutate the Escherichia coli genome, which ...
[摘要] 酶活化诱导的脱氨酶（AID）通过其将脱氧胞苷脱氨基到DNA中的脱氧尿苷的诱变活性来支持抗体亲和力成熟和同种型转换。脱氧尿苷的后续加工引发B细胞中体细胞超突变（SHM）和类型转换重组（CSR）的过程。 AID的结构功能分析需要灵敏和生物相关的方法来测量其各种活动。在这里我们描述简单但有效的方法来测量1）AID突变大肠杆菌基因组的能力，其提供其催化活性的指示; 2）AID通过补充DT40鸡B细胞系的衍生物来进行SHM的能力; 3）AID通过补充AID缺乏的原代小鼠B细胞来进行CSR的能力。三种方法的组合，伴随着AID亚细胞定位和蛋白质表达水平和稳定性的必要分析作为对照，允许AID的详细结构功能研究。
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