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Company: Fisher
Catalog#: BP231-100
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Bioorthogonal Labeling and Chemoselective Functionalization of Lung Extracellular Matrix
Author:
Date:
2021-02-20
[Abstract]  

Decellularized extracellular matrix (ECM) biomaterials derived from native tissues and organs are widely used for tissue engineering and wound repair. To boost their regenerative potential, ECM biomaterials can be functionalized via the immobilization of bioactive molecules. To enable ECM functionalization in a chemoselective manner, we have recently reported an effective approach for labeling native organ ECM with the click chemistry-reactive azide ligand via physiologic post-translational glycosylation. Here, using the rat lung as a model, we provide a detailed protocol for in vivo and ex vivo metabolic azide labeling of the native organ ECM using N-Azidoacetylgalactosamine-tetraacylated (Ac4GalNAz), together with procedures for decellularization and labeling characterization. Our

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[摘要]  [摘要]源自天然组织和器官的脱细胞细胞外基质(ECM)生物材料被广泛用于组织工程和伤口修复。为了增强其再生潜力,可以通过固定生物活性分子来使ECM生物材料功能化。为了使ECM以化学选择性的方式实现功能化,我们最近报告了一种有效的方法,可通过生理学上的翻译后糖基化,用点击化学反应的叠氮化物配体标记天然器官ECM 。在此,使用大鼠肺为模型,我们提供一种用于详细方案在体内和离体代谢叠氮化物使用N- Azidoacetylgalactosamine-tetraacylated天然器官ECM的标记(AC 4GalNAz),以及用于脱细胞和标记表征的程序。我们的方法可以在体内三天内或离体器官培养期间的一天之内进行特异性而稳定的ECM标记。脱细胞后,所得的ECM标记保持稳定。通过我们的方法,ECM生物材料可以用所需的炔烃修饰的生物分子(例如生长因子和糖胺聚糖)进行功能化,以用于组织工程和再生应用。

关键字:细胞外基质,脱细胞,生物正交,化学选择性功能化,点击化学,肺



[背景]细胞外基质(ECM)是由特定组织或器官的非细胞成分组成的水合网络支架,在通过其所包含的生物活性成分(例如纤维蛋白,生长)支持住宅细胞的活动中起关键作用。因子和糖胺聚糖(GAG)(Theocharis et ...

Ratiometric Measurement of Protein Abundance after Transient Expression of a Transgene in Nicotiana benthamiana
Author:
Date:
2020-09-05
[Abstract]  Ratiometric reporters are tools to dynamically measure the relative abundance of a protein of interest. In these systems, a target protein fused to a fluorescent or bioluminescent reporter is expressed with fixed stoichiometry to a reference protein fused to a second reporter. Both fusion proteins are encoded on a single transcript but are separated during translation by a 2A “self-cleaving” peptide. This approach enables changes in the relative abundance of a target protein to be detected sensitively, reducing variability in expression of the ratiometric reporter transgene that may occur across different tissues or transformation events. We recently developed a set of Gateway-compatible plant transformation vectors termed pRATIO that combine a variety of promoters, fluorescent and ... [摘要]  [摘要] 比率报告基因是动态测量目标蛋白质相对丰度的工具。在这些系统中,与荧光或生物发光报告基因融合的靶蛋白以固定的化学计量比与与第二报告基因融合的参考蛋白表达。两种融合蛋白均编码在单个转录本上,但在翻译过程中被2A“自切割”肽隔开。这种方法使目标蛋白质的相对丰度的变化能够被敏感地检测到,从而减少了可能在不同组织或转化事件中发生的比例报告基因转基因表达的变异性。我们最近开发了一套与网关兼容的植物转化载体,称为pRATIO 结合了多种启动子,荧光和生物发光报告基因以及源自口蹄疫病毒的2A肽。在这里,我们详细描述了如何使用双荧光比率记者pRATIO3212检查靶蛋白的瞬时表达后的相对丰度烟草本塞姆氏叶。对于此示例,我们分析了响应于karrikins 和rac -GR24 处理的拟南芥MAX2 1(SMAX1)蛋白抑制子的降解。该方案提供了一种简单,快速,易于扩展的方法,用于体内分析农杆菌浸润的烟草叶片组织中的相对蛋白质丰度。

[背景] Karrikins (KARs )是在烟雾中发现的丁烯内酯化合物,可刺激拟南芥种子萌发并增强幼苗的光形态发生(Flematti ,2004; Nelson 等,2009、2010 和2012)。拟南芥中的KAR响应需要α/β- 水解酶KARRIKIN对光的敏感性/低敏感性(KAI2 / HTL)(Sun and Ni,2011; ...

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