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Protocol to analyze chromatin-bound proteins through the cell cycle using Chromoflow flow cytometry

Chromatin-bound proteins have been conventionally measured through subcellular fractionation followed by immunoblotting or by immunofluorescence microscopy. Here, we present Chromoflow, a protocol for the quantitative analyses of protein levels on chromatin in single cells and throughout the cell cy...

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Detalles Bibliográficos
Autores principales: Alonso-Gil, Dácil, Losada, Ana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10510066/
https://www.ncbi.nlm.nih.gov/pubmed/37725510
http://dx.doi.org/10.1016/j.xpro.2023.102568
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author Alonso-Gil, Dácil
Losada, Ana
author_facet Alonso-Gil, Dácil
Losada, Ana
author_sort Alonso-Gil, Dácil
collection PubMed
description Chromatin-bound proteins have been conventionally measured through subcellular fractionation followed by immunoblotting or by immunofluorescence microscopy. Here, we present Chromoflow, a protocol for the quantitative analyses of protein levels on chromatin in single cells and throughout the cell cycle using flow cytometry. We describe steps for harvesting cells and for nuclear extraction, and a barcoding strategy to multiplex samples from different conditions that reduces antibody staining variability and eliminates the need for normalization.(1)(,)(2) We then detail procedures for data acquisition and analysis. For complete details on the use and execution of this protocol, please refer to Alonso-Gil et al. (2023).(3)
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spelling pubmed-105100662023-09-21 Protocol to analyze chromatin-bound proteins through the cell cycle using Chromoflow flow cytometry Alonso-Gil, Dácil Losada, Ana STAR Protoc Protocol Chromatin-bound proteins have been conventionally measured through subcellular fractionation followed by immunoblotting or by immunofluorescence microscopy. Here, we present Chromoflow, a protocol for the quantitative analyses of protein levels on chromatin in single cells and throughout the cell cycle using flow cytometry. We describe steps for harvesting cells and for nuclear extraction, and a barcoding strategy to multiplex samples from different conditions that reduces antibody staining variability and eliminates the need for normalization.(1)(,)(2) We then detail procedures for data acquisition and analysis. For complete details on the use and execution of this protocol, please refer to Alonso-Gil et al. (2023).(3) Elsevier 2023-09-18 /pmc/articles/PMC10510066/ /pubmed/37725510 http://dx.doi.org/10.1016/j.xpro.2023.102568 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Alonso-Gil, Dácil
Losada, Ana
Protocol to analyze chromatin-bound proteins through the cell cycle using Chromoflow flow cytometry
title Protocol to analyze chromatin-bound proteins through the cell cycle using Chromoflow flow cytometry
title_full Protocol to analyze chromatin-bound proteins through the cell cycle using Chromoflow flow cytometry
title_fullStr Protocol to analyze chromatin-bound proteins through the cell cycle using Chromoflow flow cytometry
title_full_unstemmed Protocol to analyze chromatin-bound proteins through the cell cycle using Chromoflow flow cytometry
title_short Protocol to analyze chromatin-bound proteins through the cell cycle using Chromoflow flow cytometry
title_sort protocol to analyze chromatin-bound proteins through the cell cycle using chromoflow flow cytometry
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10510066/
https://www.ncbi.nlm.nih.gov/pubmed/37725510
http://dx.doi.org/10.1016/j.xpro.2023.102568
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