Monitoring Wnt Protein Acylation Using an In Vitro Cyclo-Addition Reaction.
Monitoring Wnt Protein Acylation Using an In Vitro Cyclo-Addition Reaction.
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使用体外环加成反应监测 Wnt 蛋白酰化。
DOI：
10.1007/978-1-4939-6393-5_2
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
Lum,Lawrence
中科院分区：
文献类型：
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作者：
Tuladhar,Rubina;Yarravarapu,Nageswari;Lum,Lawrence
我们在这里描述了一种使用叠氮-炔环加成化学（点击化学）和SDS-PAGE可视化Wnt蛋白脂化状态的技术。该方案结合了使用炔基化棕榈酸酯探针的Wnt-IgG Fc融合蛋白的体内标记，但通过结合对固定在蛋白A树脂上的单步纯化的Wnt蛋白进行的二级环加成反应而偏离了传统方法。这种方法通过减少来自其他棕榈酰化蛋白质的标记的贡献以及通过提供用于基于蛋白质丰度标准化标记效率的稳健方法来减轻实验噪声。
We describe here a technique for visualizing the lipidation status of Wnt proteins using azide-alkyne cycloaddition chemistry (click chemistry) and SDS-PAGE. This protocol incorporates in vivo labeling of a Wnt-IgG Fc fusion protein using an alkynylated palmitate probe but departs from a traditional approach by incorporating a secondary cycloaddition reaction performed on single-step purified Wnt protein immobilized on protein A resin. This approach mitigates experimental noise by decreasing the contribution of labeling from other palmitoylated proteins and by providing a robust method for normalizing labeling efficiency based on protein abundance.