Sowa, S. T., Galera-Prat, A., Wazir, S., Alanen, H. I., Maksimainen, M. M., & Lehtiö, L. (2022). Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe. STAR Protocols, 3(1), 101147. https://doi.org/10.1016/j.xpro.2022.101147
Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe
|Author:||Sowa, Sven T.1; Galera-Prat, Albert1; Wazir, Sarah1;|
1Faculty for Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, 90220 Oulu, Finland
|Online Access:||PDF Full Text (PDF, 3.2 MB)|
|Persistent link:|| http://urn.fi/urn:nbn:fi-fe2022051335061
|Publish Date:|| 2022-08-09
Here, we describe a protocol to set up a screening assay for ADP-ribosyl binding proteins including proteins that possess O-glycosidase or N-glycosidase activities. The FRET-based assay measures the interaction of any ADP-ribosyl binding protein fused to CFP with a cysteine-ADP-ribosylated GAP-tag fused to YFP. Recombinant PtxS1 and PARP2 are used to mono-ADP-ribosylate and poly-ADP-ribosylate the GAP-tag. The protocol does not require specialized compounds or substrates, making it accessible and easy to adapt in any laboratory or for other proteins of interest. For complete details on the use and execution of this profile, please refer to Sowa et al. (2021).
|Type of Publication:||
A1 Journal article – refereed
|Field of Science:||
1182 Biochemistry, cell and molecular biology
This work was funded by the Jane and Aatos Erkko Foundation and by the Sigrid Jusélius Foundation. The use of the facilities of the Biocenter Oulu Structural Biology core facility, member of Biocentre Finland, Instruct-ERIC Centre Finland and FINStruct, as well as of Proteomics and Protein Analysis and Sequencing core facilities are gratefully acknowledged.
© 2022 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).