Scientific reports, 2017-08, Vol.7 (1), p.7278-11, Article 7278
2017
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Details
- Autor(en) / Beteiligte
- Titel
- Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity
- Ist Teil von
- Scientific reports, 2017-08, Vol.7 (1), p.7278-11, Article 7278
- Ort / Verlag
- England: Nature Publishing Group
- Erscheinungsjahr
- 2017
- Quelle
- Free E-Journal (出版社公開部分のみ）
- Beschreibungen/Notizen
- 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.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 2045-2322eISSN: 2045-2322DOI: 10.1038/s41598-017-07539-4Titel-ID: cdi_doaj_primary_oai_doaj_org_article_9349e82891754ac5af58d4cce70e19dc
- Format
- –
- Schlagworte
- Antibodies, Monoclonal - pharmacology, Argonaute 2 protein, Argonaute Proteins - antagonists & inhibitors, Chromatin, Chromatin Assembly and Disassembly, Chromatin Immunoprecipitation, Chromosomal Proteins, Non-Histone - antagonists & inhibitors, Chromosomal Proteins, Non-Histone - genetics, Chromosomal Proteins, Non-Histone - metabolism, CRISPR, CRISPR-Cas Systems, Enhancers, Gene Knockdown Techniques, Genetic Engineering, HEK293 Cells, High-Throughput Nucleotide Sequencing, Humans, Immunoprecipitation, Mass Spectrometry, Mass spectroscopy, miRNA, Monoclonal antibodies, Next-generation sequencing, Nuclei, Protein Binding, Scientific imaging, SWI/SNF complex, Transcription, Transcription Factors - antagonists & inhibitors, Transcription Factors - genetics, Transcription Factors - metabolism