Detection and Analysis of Proteins Modified by O-Linked N-Acetylglucosamine.
Detection and Analysis of Proteins Modified by O-Linked N-Acetylglucosamine.
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DOI：
10.1002/cpz1.129
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发表时间：
2021-05
期刊：
影响因子：
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通讯作者：
Zachara NE
中科院分区：
文献类型：
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作者：
Fahie K;Narayanan B;Zahra F;Reeves R;Fernandes SM;Hart GW;Zachara NE
O-GlcNAc是核、线粒体和细胞质蛋白的常见翻译后修饰，其调节正常生理学和细胞应激反应。O-GlcNAc循环的失调与II型糖尿病、心力衰竭、高血压和阿尔茨海默病的病因学以及心脏保护有关。这些协议涵盖了用于检测O-GlcNAc修饰的蛋白质和研究添加或去除O-GlcNAc的酶的简单而全面的技术。基本方案1：基本方案3：使用凝集素sWGA支持方案1：O-连接的糖基化的控制基本方案4：使用WGA-琼脂糖支持方案2：用氨基己糖苷酶消化蛋白替代方案1：使用半乳糖基转移酶检测被O-GlcNAc修饰的蛋白质支持方案3：半乳糖基转移酶的自身半乳糖基化支持方案4：半乳糖基转移酶活性测定基本方案5：通过β-消除和色谱法表征标记的聚糖基本方案6：检测96孔板中的O-GlcNAc基本方案7：OGT活性测定支持方案5：使O-GlcNAc转移酶脱盐基本方案8：O-GlcNAc酶活性的测定
O-GlcNAc is a common post-translational modification of nuclear, mitochondrial, and cytoplasmic proteins that regulates normal physiology and the cell stress response. Dysregulation of O-GlcNAc cycling is implicated in the etiology of type II diabetes, heart failure, hypertension, and Alzheimer’s disease, as well as cardioprotection. These protocols cover simple and comprehensive techniques for detecting proteins modified by O-GlcNAc and studying the enzymes that add or remove O-GlcNAc. Basic Protocol 1: Increasing the Stoichiometry of O-GlcNAc on Proteins Before Analysis Basic Protocol 2: Detection of Proteins Modified by O-GlcNAc Using Antibodies Basic Protocol 3: Detection of Proteins Modified by O-GlcNAc Using the Lectin sWGA Support Protocol 1: Control for O-Linked Glycosylation Basic Protocol 4: Detection and Enrichment of Proteins Using WGA-Agarose Support Protocol 2: Digestion of Proteins with Hexosaminidase Alternate Protocol 1: Detection of Proteins Modified by O-GlcNAc Using Galactosyltransferase Support Protocol 3: Autogalactosylation of Galactosyltransferase Support Protocol 4: Assay of Galactosyltransferase Activity Basic Protocol 5: Characterization of Labeled Glycans by β-Elimination and Chromatography Basic Protocol 6: Detection of O-GlcNAc in 96 well plates Basic Protocol 7: Assay for OGT Activity Support Protocol 5: Desalting the O-GlcNAc Transferase Basic Protocol 8: Assay for O-GlcNAcase Activity