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

ABSTRACT: 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).

SUBMITTER: Tan B

PROVIDER: S-EPMC7580243 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Tan Benedict B Peng Suat S Yatim Siti Maryam J M SMJM Gunaratne Jayantha J Hunziker Walter W Ludwig Alexander A

STAR protocols 20200731 2

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 c ...[more]

PMID: 33111110

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