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Method for detecting acetylated PD-L1 in cell lysates by immunoprecipitation and western blot analysis
Lysine acetylation is an important regulatory post-translational modification (PTM) that occurs sub-stoichiometrically, often representing less than 1% of the target protein. This makes studying endogenous protein acetylation extremely challenging. Recent reports suggest that several post-translatio...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9292098/ https://www.ncbi.nlm.nih.gov/pubmed/35849582 http://dx.doi.org/10.1371/journal.pone.0268887 |
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author | Middleton-Davis, Frances Davis, Ashley Middleton, Kim |
author_facet | Middleton-Davis, Frances Davis, Ashley Middleton, Kim |
author_sort | Middleton-Davis, Frances |
collection | PubMed |
description | Lysine acetylation is an important regulatory post-translational modification (PTM) that occurs sub-stoichiometrically, often representing less than 1% of the target protein. This makes studying endogenous protein acetylation extremely challenging. Recent reports suggest that several post-translational modifications (PTMs), including lysine acetylation, play a major role in the regulation the programmed cell death-ligand 1 (PD-L1), a clinically important protein target. An enrichment step is necessary to enable identification of the acetylated species by either antibody or mass spectrometry-based detection methods. This report describes a robust lab protocol for the enrichment and detection of endogenous acetylated PD-L1 protein. A recently developed acetyl lysine affinity matrix was utilized to enrich >90% of acetylated PD-L1 species, from a variety of cell lines, spanning a fourteen-fold range of target protein levels. Western blot analysis, using a highly sensitive PD-L1 antibody and optimized transfer times, was used to determine that the endogenous level of acetylated PD-L1 is in the range of 0.02–0.07% of total PD-L1. As validation, we demonstrate that acetylation levels increase to 0.11–0.17% of total PD-L1 after a 4h treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). The method described here is simple to perform in any lab equipped with tissue culture and western blot equipment. |
format | Online Article Text |
id | pubmed-9292098 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-92920982022-07-19 Method for detecting acetylated PD-L1 in cell lysates by immunoprecipitation and western blot analysis Middleton-Davis, Frances Davis, Ashley Middleton, Kim PLoS One Lab Protocol Lysine acetylation is an important regulatory post-translational modification (PTM) that occurs sub-stoichiometrically, often representing less than 1% of the target protein. This makes studying endogenous protein acetylation extremely challenging. Recent reports suggest that several post-translational modifications (PTMs), including lysine acetylation, play a major role in the regulation the programmed cell death-ligand 1 (PD-L1), a clinically important protein target. An enrichment step is necessary to enable identification of the acetylated species by either antibody or mass spectrometry-based detection methods. This report describes a robust lab protocol for the enrichment and detection of endogenous acetylated PD-L1 protein. A recently developed acetyl lysine affinity matrix was utilized to enrich >90% of acetylated PD-L1 species, from a variety of cell lines, spanning a fourteen-fold range of target protein levels. Western blot analysis, using a highly sensitive PD-L1 antibody and optimized transfer times, was used to determine that the endogenous level of acetylated PD-L1 is in the range of 0.02–0.07% of total PD-L1. As validation, we demonstrate that acetylation levels increase to 0.11–0.17% of total PD-L1 after a 4h treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). The method described here is simple to perform in any lab equipped with tissue culture and western blot equipment. Public Library of Science 2022-07-18 /pmc/articles/PMC9292098/ /pubmed/35849582 http://dx.doi.org/10.1371/journal.pone.0268887 Text en © 2022 Middleton-Davis et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Lab Protocol Middleton-Davis, Frances Davis, Ashley Middleton, Kim Method for detecting acetylated PD-L1 in cell lysates by immunoprecipitation and western blot analysis |
title | Method for detecting acetylated PD-L1 in cell lysates by immunoprecipitation and western blot analysis |
title_full | Method for detecting acetylated PD-L1 in cell lysates by immunoprecipitation and western blot analysis |
title_fullStr | Method for detecting acetylated PD-L1 in cell lysates by immunoprecipitation and western blot analysis |
title_full_unstemmed | Method for detecting acetylated PD-L1 in cell lysates by immunoprecipitation and western blot analysis |
title_short | Method for detecting acetylated PD-L1 in cell lysates by immunoprecipitation and western blot analysis |
title_sort | method for detecting acetylated pd-l1 in cell lysates by immunoprecipitation and western blot analysis |
topic | Lab Protocol |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9292098/ https://www.ncbi.nlm.nih.gov/pubmed/35849582 http://dx.doi.org/10.1371/journal.pone.0268887 |
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