Riham Ayoubi, Joel Ryan, Sara Gonzalez Bolivar, Charles Alende, Vera Ruiz Moleon, Maryam Fotouhi, Mona Alqazzaz, Kathleen Southern, Walaa Alshafie, Matt R Baker, Alexander R Ball, Danielle Callahan, Jeffery A Cooper, Katherine Crosby, Kevin J Harvey, Douglas W Houston, Ravindran Kumaran, Meghan Rego, Christine Schofield, Hai Wu, Michael S Biddle, Claire M Brown, Richard A Kahn, Anita Bandrowski, Harvinder S Virk, Aled M Edwards, Peter S McPherson, Carl Laflamme
{"title":"A consensus platform for antibody characterization.","authors":"Riham Ayoubi, Joel Ryan, Sara Gonzalez Bolivar, Charles Alende, Vera Ruiz Moleon, Maryam Fotouhi, Mona Alqazzaz, Kathleen Southern, Walaa Alshafie, Matt R Baker, Alexander R Ball, Danielle Callahan, Jeffery A Cooper, Katherine Crosby, Kevin J Harvey, Douglas W Houston, Ravindran Kumaran, Meghan Rego, Christine Schofield, Hai Wu, Michael S Biddle, Claire M Brown, Richard A Kahn, Anita Bandrowski, Harvinder S Virk, Aled M Edwards, Peter S McPherson, Carl Laflamme","doi":"10.1038/s41596-024-01095-8","DOIUrl":null,"url":null,"abstract":"<p><p>Antibody-based research applications are critical for biological discovery. Yet there are no industry standards for comparing the performance of antibodies in various applications. We describe a knockout cell line-based antibody characterization platform, developed and approved jointly by industry and academic researchers, that enables the systematic comparison of antibody performance in western blot, immunoprecipitation and immunofluorescence. The scalable protocols, which require minimal technological resources, consist of (1) the identification of appropriate cell lines for antibody characterization studies, (2) development/contribution of isogenic knockout controls, and (3) a series of antibody characterization procedures focused on the most common applications of antibodies in research. We provide examples of expected outcomes to guide antibody users in evaluating antibody performance. Central to our approach is advocating for transparent and open data sharing, enabling a community effort to identify specific antibodies for all human proteins. Mid-level graduate students with training in biochemistry and prior experience in cell culture and microscopy can complete the protocols for a specific protein within 1 month while working part-time on this effort. Antibody characterization is needed to meet standards for resource validation and data reproducibility, which are increasingly required by journals and funding agencies.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1000,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Nature Protocols","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1038/s41596-024-01095-8","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Antibody-based research applications are critical for biological discovery. Yet there are no industry standards for comparing the performance of antibodies in various applications. We describe a knockout cell line-based antibody characterization platform, developed and approved jointly by industry and academic researchers, that enables the systematic comparison of antibody performance in western blot, immunoprecipitation and immunofluorescence. The scalable protocols, which require minimal technological resources, consist of (1) the identification of appropriate cell lines for antibody characterization studies, (2) development/contribution of isogenic knockout controls, and (3) a series of antibody characterization procedures focused on the most common applications of antibodies in research. We provide examples of expected outcomes to guide antibody users in evaluating antibody performance. Central to our approach is advocating for transparent and open data sharing, enabling a community effort to identify specific antibodies for all human proteins. Mid-level graduate students with training in biochemistry and prior experience in cell culture and microscopy can complete the protocols for a specific protein within 1 month while working part-time on this effort. Antibody characterization is needed to meet standards for resource validation and data reproducibility, which are increasingly required by journals and funding agencies.
期刊介绍：
Nature Protocols focuses on publishing protocols used to address significant biological and biomedical science research questions, including methods grounded in physics and chemistry with practical applications to biological problems. The journal caters to a primary audience of research scientists and, as such, exclusively publishes protocols with research applications. Protocols primarily aimed at influencing patient management and treatment decisions are not featured.
The specific techniques covered encompass a wide range, including but not limited to: Biochemistry, Cell biology, Cell culture, Chemical modification, Computational biology, Developmental biology, Epigenomics, Genetic analysis, Genetic modification, Genomics, Imaging, Immunology, Isolation, purification, and separation, Lipidomics, Metabolomics, Microbiology, Model organisms, Nanotechnology, Neuroscience, Nucleic-acid-based molecular biology, Pharmacology, Plant biology, Protein analysis, Proteomics, Spectroscopy, Structural biology, Synthetic chemistry, Tissue culture, Toxicology, and Virology.
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