Protocol to study direct (de)phosphorylation events on a proteome-wide scale using on-bead in vitro enzyme assays
DOKPE
- Melanie, Brunner University of Fribourg
- Zehan, Hu University of Fribourg
- Marco , Caligaris University of Fribourg
- Michael, Stumpe University of Fribourg
- Claudio, De Virgilio University of Fribourg
- Joern, Dengjel ORCID University of Fribourg
- 2026
Published in:
- STAR Protocols. - Elsevier BV. - 2026, vol. 7, no. 3, p. 104629
English
Protein phosphorylation regulates essential cellular processes, yet probing ki nase and phosphatase activities in native contexts remains challenging. Here, we present a protocol to study direct (de)phosphorylation events on a prote-ome-wide scale using on-bead in vitro kinase and phosphatase assays (OBIKA/
OBIPhA). We describe steps for employing active enzyme complexes and identi
fying direct substrates and regulated phosphosites using mass spectrometry.
Compared to traditional in vitro approaches, this protocol operates under native
conditions to preserve protein interactions and modifications in eukaryotic cell
systems.
For complete details on the use and execution of this protocol, please refer to
Brunner et al. 1 and Hu et al. 2
OBIPhA). We describe steps for employing active enzyme complexes and identi
fying direct substrates and regulated phosphosites using mass spectrometry.
Compared to traditional in vitro approaches, this protocol operates under native
conditions to preserve protein interactions and modifications in eukaryotic cell
systems.
For complete details on the use and execution of this protocol, please refer to
Brunner et al. 1 and Hu et al. 2
- Faculty
- Faculté des sciences et de médecine
- Department
- Département de Biologie
- Language
-
- English
- Classification
- Biological sciences
- Other electronic version
- License
-
CC BY
- Open access status
- gold
- Identifiers
-
- DOI 10.1016/j.xpro.2026.104629
- ISSN 2666-1667
- Persistent URL
- https://folia.unifr.ch/unifr/documents/335871
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