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On-Slide Lambda Protein Phosphatase-Mediated Dephosphorylation of Fixed Samples

Protein phosphorylation is a ubiquitous post-translational modification that regulates a plethora of intracellular processes, making its analysis crucial for understanding intracellular dynamics. The commonly used methods, such as radioactive labeling and gel electrophoresis, do not provide informat...

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Autores principales: Tishchenko, Alexander, Van Waesberghe, Cliff, Favoreel, Herman W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10304472/
https://www.ncbi.nlm.nih.gov/pubmed/37367999
http://dx.doi.org/10.3390/mps6030055
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author Tishchenko, Alexander
Van Waesberghe, Cliff
Favoreel, Herman W.
author_facet Tishchenko, Alexander
Van Waesberghe, Cliff
Favoreel, Herman W.
author_sort Tishchenko, Alexander
collection PubMed
description Protein phosphorylation is a ubiquitous post-translational modification that regulates a plethora of intracellular processes, making its analysis crucial for understanding intracellular dynamics. The commonly used methods, such as radioactive labeling and gel electrophoresis, do not provide information about subcellular localization. Immunofluorescence using phospho-specific antibodies and subsequent analysis via microscopy allows researchers to assess subcellular localization, but it typically lacks validation whether the observed fluorescent signal is phosphorylation specific. In this study, an on-slide dephosphorylation assay coupled with immunofluorescence staining using phospho-specific antibodies on fixed samples is proposed as a fast and simple approach to validate phosphorylated proteins in their native subcellular context. The assay was validated using antibodies against two different phosphorylated target proteins, connexin 43 phosphorylated at serine 373, and phosphorylated substrates of protein kinase A, with a dramatic reduction in the signal upon dephosphorylation. The proposed approach provides a convenient way to validate phosphorylated proteins without the need for additional sample preparation steps, reducing the time and effort required for analysis, while minimizing the risk of protein loss or alteration.
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spelling pubmed-103044722023-06-29 On-Slide Lambda Protein Phosphatase-Mediated Dephosphorylation of Fixed Samples Tishchenko, Alexander Van Waesberghe, Cliff Favoreel, Herman W. Methods Protoc Technical Note Protein phosphorylation is a ubiquitous post-translational modification that regulates a plethora of intracellular processes, making its analysis crucial for understanding intracellular dynamics. The commonly used methods, such as radioactive labeling and gel electrophoresis, do not provide information about subcellular localization. Immunofluorescence using phospho-specific antibodies and subsequent analysis via microscopy allows researchers to assess subcellular localization, but it typically lacks validation whether the observed fluorescent signal is phosphorylation specific. In this study, an on-slide dephosphorylation assay coupled with immunofluorescence staining using phospho-specific antibodies on fixed samples is proposed as a fast and simple approach to validate phosphorylated proteins in their native subcellular context. The assay was validated using antibodies against two different phosphorylated target proteins, connexin 43 phosphorylated at serine 373, and phosphorylated substrates of protein kinase A, with a dramatic reduction in the signal upon dephosphorylation. The proposed approach provides a convenient way to validate phosphorylated proteins without the need for additional sample preparation steps, reducing the time and effort required for analysis, while minimizing the risk of protein loss or alteration. MDPI 2023-05-27 /pmc/articles/PMC10304472/ /pubmed/37367999 http://dx.doi.org/10.3390/mps6030055 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Technical Note
Tishchenko, Alexander
Van Waesberghe, Cliff
Favoreel, Herman W.
On-Slide Lambda Protein Phosphatase-Mediated Dephosphorylation of Fixed Samples
title On-Slide Lambda Protein Phosphatase-Mediated Dephosphorylation of Fixed Samples
title_full On-Slide Lambda Protein Phosphatase-Mediated Dephosphorylation of Fixed Samples
title_fullStr On-Slide Lambda Protein Phosphatase-Mediated Dephosphorylation of Fixed Samples
title_full_unstemmed On-Slide Lambda Protein Phosphatase-Mediated Dephosphorylation of Fixed Samples
title_short On-Slide Lambda Protein Phosphatase-Mediated Dephosphorylation of Fixed Samples
title_sort on-slide lambda protein phosphatase-mediated dephosphorylation of fixed samples
topic Technical Note
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10304472/
https://www.ncbi.nlm.nih.gov/pubmed/37367999
http://dx.doi.org/10.3390/mps6030055
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