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Enhanced validation of antibodies for research applications.

ABSTRACT: There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.

SUBMITTER: Edfors F

PROVIDER: S-EPMC6175901 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Enhanced validation of antibodies for research applications.

Edfors Fredrik F Hober Andreas A Linderbäck Klas K Maddalo Gianluca G Azimi Alireza A Sivertsson Åsa Å Tegel Hanna H Hober Sophia S Szigyarto Cristina Al-Khalili CA Fagerberg Linn L von Feilitzen Kalle K Oksvold Per P Lindskog Cecilia C Forsström Björn B Uhlen Mathias M

Nature communications 20181008 1

There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strat ...[more]

PMID: 30297845

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Enhanced validation of antibodies for research applications

Project description:There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation “pillars" were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies and migration capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.

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Enhanced Validation of Antibodies Enables the Discovery of Missing Proteins.

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Project description:Summary Cytotoxic natural killer cells kill tumors and infected cells. We carried out CRISPR-based gene editing and transcriptional regulation in hard-to-manipulate NK-92 cells. NK-92-based therapies were found to be safe and efficacious in preclinical studies of cancers. Here, we have pioneered the generation and validation of NK-92 cells constitutively expressing Cas9 or dCas9 for knockout (CRISPRko), transcriptional activation (CRISPRa), or transcriptional repression (CRISPRi) of genes. Our CRISPR-engineered NK-92 cell platforms can be modified for research and off-the-shelf therapeutic applications. Graphical abstract Highlights • Constitutively repressing or activating genes in human NK cells is difficult• CRISPR-engineered natural killer cells are attractive for therapeutic applications• Our approach generates stable CRISPRko, CRISPRi, and CRISPRa human NK cell lines Cytotoxic natural killer cells kill tumors and infected cells. We carried out CRISPR-based gene editing and transcriptional regulation in hard-to-manipulate NK-92 cells. NK-92-based therapies were found to be safe and efficacious in preclinical studies of cancers. Here, we have pioneered the generation and validation of NK-92 cells constitutively expressing Cas9 or dCas9 for knockout (CRISPRko), transcriptional activation (CRISPRa), or transcriptional repression (CRISPRi) of genes. Our CRISPR-engineered NK-92 cell platforms can be modified for research and off-the-shelf therapeutic applications.

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