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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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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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. |
format | Online Article Text |
id | pubmed-10304472 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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