Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe

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 f...

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Detalles Bibliográficos
Autores principales: Sowa, Sven T., Galera-Prat, Albert, Wazir, Sarah, Alanen, Heli I., Maksimainen, Mirko M., Lehtiö, Lari
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8810561/
https://www.ncbi.nlm.nih.gov/pubmed/35141567
http://dx.doi.org/10.1016/j.xpro.2022.101147
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author Sowa, Sven T.
Galera-Prat, Albert
Wazir, Sarah
Alanen, Heli I.
Maksimainen, Mirko M.
Lehtiö, Lari
author_facet Sowa, Sven T.
Galera-Prat, Albert
Wazir, Sarah
Alanen, Heli I.
Maksimainen, Mirko M.
Lehtiö, Lari
author_sort Sowa, Sven T.
collection PubMed
description 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).
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spelling pubmed-88105612022-02-08 Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe Sowa, Sven T. Galera-Prat, Albert Wazir, Sarah Alanen, Heli I. Maksimainen, Mirko M. Lehtiö, Lari STAR Protoc Protocol 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). Elsevier 2022-01-29 /pmc/articles/PMC8810561/ /pubmed/35141567 http://dx.doi.org/10.1016/j.xpro.2022.101147 Text en © 2022 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Sowa, Sven T.
Galera-Prat, Albert
Wazir, Sarah
Alanen, Heli I.
Maksimainen, Mirko M.
Lehtiö, Lari
Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe
title Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe
title_full Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe
title_fullStr Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe
title_full_unstemmed Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe
title_short Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe
title_sort preparation of screening assays for adp-ribosyl readers and erasers using the gap-tag as a binding probe
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8810561/
https://www.ncbi.nlm.nih.gov/pubmed/35141567
http://dx.doi.org/10.1016/j.xpro.2022.101147
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