Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity

Abstract In this study, we originally aimed to characterize the potential role of Argonaute 2 (AGO2) in the nucleus, a key protein of the miRNA machinery. We combined Chromatin Immunoprecipitation (ChIP) with high throughput sequencing (ChIP-seq) and quantitative mass spectrometry (ChIP-MS) using th...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Roderick A. P. M. van Eijl, Teun van den Brand, Luan N. Nguyen, Klaas W. Mulder
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9349e82891754ac5af58d4cce70e19dc
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:9349e82891754ac5af58d4cce70e19dc
record_format	dspace
spelling	oai:doaj.org-article:9349e82891754ac5af58d4cce70e19dc2021-12-02T15:05:19ZReactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity10.1038/s41598-017-07539-42045-2322https://doaj.org/article/9349e82891754ac5af58d4cce70e19dc2017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-07539-4https://doaj.org/toc/2045-2322Abstract In this study, we originally aimed to characterize the potential role of Argonaute 2 (AGO2) in the nucleus, a key protein of the miRNA machinery. We combined Chromatin Immunoprecipitation (ChIP) with high throughput sequencing (ChIP-seq) and quantitative mass spectrometry (ChIP-MS) using the broadly used AGO2 11A9 antibody to determine interactions with chromatin and nuclear proteins. We found a previously described interaction between AGO2 and SWI/SNF on chromatin with ChIP-MS and observed enrichment at enhancers and transcription start sites using ChIP-seq. However, antibody specificity issues can produce misleading results for ChIP, RNA-seq and Mass spectrometry. Therefore, we developed a CRISPR/Cas9 engineered AGO2−/− HEK293T cell line to validate our findings. ChIP-qPCR and immunoprecipitation combined with MS (IP-MS) showed that the 11A9 antibody associates with chromatin and SWI/SNF in the absence of AGO2. Furthermore, stoichiometry, IP-MS and co-IP analysis suggests a direct interaction of this antibody with SMARCC1, a component of the SWI/SNF complex. For this reason, particular care should be taken in performing and interpreting experiments in which the 11A9 antibody is used to study a nuclear role of AGO2.Roderick A. P. M. van EijlTeun van den BrandLuan N. NguyenKlaas W. MulderNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Roderick A. P. M. van Eijl Teun van den Brand Luan N. Nguyen Klaas W. Mulder Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity
description	Abstract In this study, we originally aimed to characterize the potential role of Argonaute 2 (AGO2) in the nucleus, a key protein of the miRNA machinery. We combined Chromatin Immunoprecipitation (ChIP) with high throughput sequencing (ChIP-seq) and quantitative mass spectrometry (ChIP-MS) using the broadly used AGO2 11A9 antibody to determine interactions with chromatin and nuclear proteins. We found a previously described interaction between AGO2 and SWI/SNF on chromatin with ChIP-MS and observed enrichment at enhancers and transcription start sites using ChIP-seq. However, antibody specificity issues can produce misleading results for ChIP, RNA-seq and Mass spectrometry. Therefore, we developed a CRISPR/Cas9 engineered AGO2−/− HEK293T cell line to validate our findings. ChIP-qPCR and immunoprecipitation combined with MS (IP-MS) showed that the 11A9 antibody associates with chromatin and SWI/SNF in the absence of AGO2. Furthermore, stoichiometry, IP-MS and co-IP analysis suggests a direct interaction of this antibody with SMARCC1, a component of the SWI/SNF complex. For this reason, particular care should be taken in performing and interpreting experiments in which the 11A9 antibody is used to study a nuclear role of AGO2.
format	article
author	Roderick A. P. M. van Eijl Teun van den Brand Luan N. Nguyen Klaas W. Mulder
author_facet	Roderick A. P. M. van Eijl Teun van den Brand Luan N. Nguyen Klaas W. Mulder
author_sort	Roderick A. P. M. van Eijl
title	Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity
title_short	Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity
title_full	Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity
title_fullStr	Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity
title_full_unstemmed	Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity
title_sort	reactivity of human ago2 monoclonal antibody 11a9 with the swi/snf complex: a case study for rigorously defining antibody selectivity
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/9349e82891754ac5af58d4cce70e19dc
work_keys_str_mv	AT roderickapmvaneijl reactivityofhumanago2monoclonalantibody11a9withtheswisnfcomplexacasestudyforrigorouslydefiningantibodyselectivity AT teunvandenbrand reactivityofhumanago2monoclonalantibody11a9withtheswisnfcomplexacasestudyforrigorouslydefiningantibodyselectivity AT luannnguyen reactivityofhumanago2monoclonalantibody11a9withtheswisnfcomplexacasestudyforrigorouslydefiningantibodyselectivity AT klaaswmulder reactivityofhumanago2monoclonalantibody11a9withtheswisnfcomplexacasestudyforrigorouslydefiningantibodyselectivity
_version_	1718388906320199680

Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity

Ejemplares similares