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Glycogen (5 mg/ml)

Glycogen (5 mg/ml)

Company: Thermo Fisher Scientific
Catalog#: AM9510
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Coupling Exonuclease Digestion with Selective Chemical Labeling for Base-resolution Mapping of 5-Hydroxymethylcytosine in Genomic DNA
Author:
Date:
2018-03-05
[Abstract]  This protocol is designed to obtain base-resolution information on the level of 5-hydroxymethylcytosine (5hmC) in CpGs without the need for bisulfite modification. It relies on (i) the capture of hydroxymethylated sequences by a procedure known as ‘selective chemical labeling’ (see Szulwach et al., 2012) and (ii) the digestion of the captured DNA by exonucleases. After Illumina sequencing of the digested DNA fragments, an ad hoc bioinformatic pipeline extracts the information for further downstream analysis. [摘要]  该协议旨在获得CpGs中5-羟甲基胞嘧啶(5hmC)水平的碱基分辨率信息,而无需亚硫酸氢盐修饰。 它依赖于(i)通过称为“选择性化学标记”(参见Szulwach等人,2012)的方法捕获羟甲基化序列和(ii)通过外切核酸酶消化捕获的DNA。 在消化的DNA片段的Illumina测序之后,特设的生物信息学管道提取信息用于进一步的下游分析。

【背景】基因组DNA中胞嘧啶的甲基化可以被蛋白质读取,并且主要被翻译成基因沉默。基因组中的大多数CpG二核苷酸是甲基化的,包括位于基因调控区如增强子的那些。然而,当需要时,这些CpG可以通过Ten Eleven Translocation(TET)酶将甲基氧化并且通过碱基切除修复系统用未甲基化的胞嘧啶置换来去甲基化。 5-羟甲基胞嘧啶(5hmC)是5-甲基胞嘧啶的第一个氧化衍生物,并且在基因组中绘制该修饰的碱基提供了关于正在进行活性去甲基化的区域的信息。尽管选择性化学标记(SCL)可以非常特异地检测5hmC,但该技术的分辨率受DNA片段大小的限制,特别是当捕获的DNA中存在多个CpG时。为了提高分辨率,我们引入了使用外切核酸酶的消化步骤,所述核酸外切酶将DNA分子修剪成靠近羟甲基化的胞嘧啶(Sérandour et。,2016)。然后对测序读数进行适当的生物信息学处理,然后将羟甲基化评分赋予捕获的CpG。

Intracellular Glycogen Assays
Author:
Date:
2014-06-05
[Abstract]  Glycogen, a soluble multi-branched glucose homopolysaccharide, is composed of chains of α-1,4-linked glucose residues interconnected by α-1,6-linked branches. The classical biosynthetic pathway involves phosphoglucomutase (Pgm), glucose-1-phosphate adenylyltransferase (GlgC or GlgCD), glycogen synthase (GlgA) and branching enzyme (GlgB). Phosphoglucomutase converts glucose-6-phosphate into glucose-1-phosphate, which serves as a substrate for ADP-glucose synthesis catalyzed by GlgC or GlgCD. Then, GlgA catalyzes the transfer of glucosyl units from ADP-glucose to the elongating chain of linear α-1,4-glucan. GlgB subsequently cleaves off portions of the glucan and links it to internal glucose molecules in existing chains via α-1,6 glycosidic bonds to form the glycogen structure. Glycogen ... [摘要]  糖原,一种可溶性多分支葡萄糖同多糖,由通过α-1,6连接的分支相互连接的α-1,4-连接的葡萄糖残基的链组成。经典的生物合成途径包括磷酸葡萄糖变位酶(Pgm),葡萄糖-1-磷酸腺苷酰转移酶(GlgC或GlgCD),糖原合酶(GlgA)和分支酶(GlgB)。磷酸葡萄糖变位酶将葡萄糖-6-磷酸转化为葡萄糖-1-磷酸,其作为由GlgC或GlgCD催化的ADP-葡萄糖合成的底物。然后,GlgA催化葡萄糖基单位从ADP-葡萄糖向线性α-1,4-葡聚糖的延长链的转移。 GlgB随后切割葡聚糖的部分并且通过α-1,6糖苷键将其连接到现有链中的内部葡萄糖分子以形成糖原结构。糖原分解由糖原磷酸化酶(GlgP)和脱支酶(GlgX)介导,其催化来自非还原末端的葡聚糖链中的α-1,4-葡萄糖基键的顺序磷酸解和由GlgP产生的极限糊精的脱支, 分别。越来越多的研究已经揭示糖原代谢在一些原核生物的多种生理功能中的参与超过合成能量储存化合物的功能。嗜酸乳杆菌 NCFM是第一种证明具有功能性糖原生物合成途径的益生菌乳酸菌,其参与其生长,胆汁耐受性和复杂的碳水化合物代谢(Goh和Klaenhammer,2013)。用于嗜酸乳杆菌(嗜酸乳杆菌)的以下定性(用于检测细胞内糖原)和定量(用于测量细胞内糖原含量)细胞内糖原测定方案从之前的工作(Govons等人,1969; Law等人,1995; ...

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