An Optimized Protocol for Proximity Biotinylation in Confluent Epithelial Cell Cultures Using the Peroxidase APEX2

• Published in: STAR protocols 2020-09, Vol.1 (2), p.100074-100074, Article 100074
• Main Authors: Tan, Benedict, Peng, Suat, Yatim, Siti Maryam J.M., Gunaratne, Jayantha, Hunziker, Walter, Ludwig, Alexander
• Format: Article
• Language: English
• Subjects: Animals; Biotinylation - methods; Cell Culture Techniques - methods; Dogs; Epithelial Cells - cytology; Madin Darby Canine Kidney Cells; Peroxidase - metabolism; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2020.100074

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). [Display omitted] •APEX2 permits proximity biotinylation in confluent cell cultures•Biotin phenol and peroxide concentrations are critical•Spatial controls are required to generate specific proximity proteomes The peroxidase APEX2 has been widely used 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.