Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 2 de 2
Filtrar
Mais filtros

Bases de dados
Ano de publicação
Tipo de documento
País de afiliação
Intervalo de ano de publicação
1.
Nat Methods ; 15(5): 330-338, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29638227

RESUMO

A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.


Assuntos
Anticorpos Monoclonais/imunologia , Análise Serial de Proteínas/métodos , Fatores de Transcrição/metabolismo , Animais , Clonagem Molecular , Bases de Dados Factuais , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes
2.
Mol Cell Proteomics ; 11(6): O111.016253, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22307071

RESUMO

To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His(6) fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Especificidade de Anticorpos , Proteoma/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Antígenos/química , Antígenos/imunologia , Linhagem Celular Tumoral , Humanos , Hibridomas , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Análise Serial de Proteínas , Proteoma/química , Proteínas Recombinantes de Fusão/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA