自噬
蛋白质水解
细胞生物学
生物
流式细胞术
焊剂(冶金)
荧光显微镜
绿色荧光蛋白
荧光
分子生物学
生物物理学
生物化学
酶
化学
细胞凋亡
基因
物理
有机化学
量子力学
作者
Willa Wen-You Yim,Hayashi Yamamoto,Noboru Mizushima
出处
期刊:Autophagy
[Informa]
日期:2022-09-19
卷期号:19 (4): 1363-1364
标识
DOI:10.1080/15548627.2022.2123638
摘要
Monitoring mammalian macroautophagic/autophagic flux is necessary in most autophagy studies but has generally been difficult to do. Here, we discuss our recent report of a HaloTag-based processing method that offers a straightforward readout for autophagic flux. We found that the self-labeling protein HaloTag becomes resistant to proteolysis when labeled with its ligand. Fusing HaloTag to an autophagy protein such as LC3 results in a reporter that is completely degraded when delivered into lysosomes but, when pulse-labeled with HaloTag ligand, releases free HaloTagligand when processed by lysosomal enzymes. The quantifiable amount of free HaloTagligand, observed by immunoblotting or in-gel fluorescence detection, reflects autophagic flux. Besides being compatible with fluorescence microscopy and flow cytometry applications, this quantitative assay can be readily adapted to monitor most autophagy pathways or the autophagic degradation of a protein of interest.
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