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

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.

Fluorescence ImmunoPrecipitation (FLIP): a Novel Assay for High-Throughput IP.

Background: The immunoprecipitation (IP) assay is a valuable molecular biology tool applied across a breadth of fields. The standard assay couples IP to immunoblotting (IP/IB), a procedure severely limited as it is not easily scaled for high-throughput analysis.

Results: Here we describe and characterize a new methodology for fast and reliable evaluation of an immunoprecipitation reaction.

Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach.

Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion.

Rapid identification of monospecific monoclonal antibodies using a human proteome microarray.

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.

Analysis of commercial Entamoeba histolytica ELISA kits for the detection of Entamoeba invadens in reptiles.

Entamoeba invadens is pathogenic in multiple reptile species and has caused severe outbreaks in zoos and other facilities worldwide. Infections can be difficult to diagnose and to differentiate from other reptilian Entamoeba species. The goal of this study was to determine if kits developed to identify the human pathogen Entamoeba histolytica could be used to detect E.

Trichostatin A effects on gene expression in the protozoan parasite Entamoeba histolytica.

Background: Histone modification regulates chromatin structure and influences gene expression associated with diverse biological functions including cellular differentiation, cancer, maintenance of genome architecture, and pathogen virulence. In Entamoeba, a deep-branching eukaryote, short chain fatty acids (SCFA) affect histone acetylation and parasite development. Additionally, a number of active histone modifying enzymes have been identified in the parasite genome.

Identification of developmentally regulated genes in Entamoeba histolytica: insights into mechanisms of stage conversion in a protozoan parasite.

Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. The protozoan parasite Entamoeba histolytica converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Identification of genes involved in the developmental pathway has been severely hindered by the inability to generate E.