Dataset Information

A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors.

ABSTRACT: 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.

SUBMITTER: Venkataraman A

PROVIDER: S-EPMC6063793 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors.

Venkataraman Anand A Yang Kun K Irizarry Jose J Mackiewicz Mark M Mita Paolo P Kuang Zheng Z Xue Lin L Ghosh Devlina D Liu Shuang S Ramos Pedro P Hu Shaohui S Bayron Kain Diane D Keegan Sarah S Saul Richard R Colantonio Simona S Zhang Hongyan H Behn Florencia Pauli FP Song Guang G Albino Edisa E Asencio Lillyann L Ramos Leonardo L Lugo Luvir L Morell Gloriner G Rivera Javier J Ruiz Kimberly K Almodovar Ruth R Nazario Luis L Murphy Keven K Vargas Ivan I Rivera-Pacheco Zully Ann ZA Rosa Christian C Vargas Moises M McDade Jessica J Clark Brian S BS Yoo Sooyeon S Khambadkone Seva G SG de Melo Jimmy J Stevanovic Milanka M Jiang Lizhi L Li Yana Y Yap Wendy Y WY Jones Brittany B Tandon Atul A Campbell Elliot E Montelione Gaetano T GT Anderson Stephen S Myers Richard M RM Boeke Jef D JD Fenyö David D Whiteley Gordon G Bader Joel S JS Pino Ignacio I Eichinger Daniel J DJ Zhu Heng H Blackshaw Seth S

Nature methods 20180319 5

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. ...[more]

PMID: 29638227

Similar Datasets

Project description:Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28.Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell.

Dataset Information

A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors.

Publications

A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets