Project description:The aim of the study was to determine the epitope targeted by 31E10/E7 mouse monoclonal antibody (mAb) and the cross-reactivity to linear peptide epitopes of 10 different Neisserial Heparin Binding Antigen (NHBA) variants. mAb 31E10/E7 was diluted at 1:200 and incubated on a custom PepStar Peptide Microarray platform printed with 560 different peptides in three independent experiments.
Project description:In this paper several computer programs were used to simulate in situ synthesis of peptides using shadow masks and BOC synthesis. The peptides were designed to be random, or pseudo-random, but fulfill requirements of immunosignaturing. This file contains data from actual 330,000 peptide arrays that used the first iteration of the peptide generation algorithm. Monoclonal antibodies were bound to the microarrays and the total number of peptides that distinguished each monoclonal was measured. This provides a baseline against which to compare purely random sequences. One replicate of each peptide was printed on 1 330k peptide microarray. One microarray were tested for each sample. Image was qualified using in-house metrics for quality assurance.
Project description:The study of chromatin and its organization involves use of antibodies directed against thousands of distinct proteins spanning numerous isoforms and post-translationally modified variants in the human nucleus. Antibodies are essential where epitope-tagging is impractical (e.g., human tissue, cell lines from a wide variety of origins). However, antibody reliability is problematic. The Protein Capture Reagents Program (PCRP) has generated about a thousand renewable monoclonal antibodies that are expected to be more reliable, but these reagents have not been field tested. Since they were generated primarily against chromatin proteins, we tested them in a variety of chromatin-based assays. 887 unique antibodies against 681 unique chromatin targets were assayed by ChIP-exo. A subset were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and PBM protein-DNA interaction assays. While this represents only a small portion of the antibody validation/utility space, and noting that optimization may be needed in each assay, our findings suggest that 10-20% of the PCRP-derived antibodies have utility in a particular biochemical assay. We also describe validation strategies.
Project description:Antibodies offer a powerful means to interrogate specific proteins in a complex milieu, and where epitope tagging is impractical. However, antibody availability and reliability are problematic. The Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies against human-presumptive chromatin proteins in an effort to improve reliability. However, these reagents have not been widely field-tested. We therefore screened their ability in a variety of assays. 887 unique antibodies against 681 unique chromatin proteins, of which 605 are putative sequence-specific transcription factors (TFs), were assayed by ChIP-exo. Subsets were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. At least 6% of the tested antibodies were validated for use in ChIP-based assays by the most stringent of our criteria. An additional 34% produced data suggesting they warranted further testing for clearer validation. We demonstrate and discuss the metrics and limitations to antibody validation in chromatin-based assays.
