Epithelial Cell Biology Laboratory, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A∗STAR), 61 Biopolis Drive, Singapore 138673, Singapore.
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 2 Medical Drive, Singapore 117593, Singapore.
STAR Protoc. 2020 Jul 31;1(2):100074. doi: 10.1016/j.xpro.2020.100074. eCollection 2020 Sep 18.
The peroxidase APEX2 has been used widely for proximity biotinylation and subsequent proteomics analyses. However, the poor membrane permeability of the biotin phenol substrate and the inhibitory effect of peroxide on the enzyme's activity has hampered proximity labeling in certain cell culture systems and tissues. Here, we describe an APEX2 protocol that uses alternative peroxide and biotin phenol concentrations. The protocol permits robust proximity biotinylation in confluent epithelial cell cultures and may be applicable to other cell cultures and tissues. For complete details on the use and execution of this protocol, please refer to Tan et al. (2020).
过氧化物酶 APEX2 已被广泛用于邻近生物素化和随后的蛋白质组学分析。然而，生物素酚底物的膜通透性差以及过氧化物对酶活性的抑制作用，阻碍了某些细胞培养系统和组织中的邻近标记。在这里，我们描述了一种使用替代过氧化物和生物素酚浓度的 APEX2 方案。该方案允许在致密上皮细胞培养物中进行强大的邻近生物素化，并且可能适用于其他细胞培养物和组织。有关此协议的使用和执行的完整详细信息，请参阅 Tan 等人。(2020)。
