Project description:We demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by LC-MS/MS in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron transfer high-energy collision dissociation (EThcD) on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody Herceptin, with an accuracy of 98% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of KYP in 10 days old KYPpro::KYP:3xFLAG arabidopsis. By obtaining sequence from chromatin immunoprecipitated DNA, we mapped genome-wide binding levels of KYP:3xFLAG. ChIP was performed using anti-FLAG antibody (sigma M2), and ChIP DNA were analyzed by Illumina NovoSeq.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of HDLpro::HDL-YFP in 10 days old KYPpro::KYP:3xFLAG arabidopsis. By obtaining sequence from chromatin immunoprecipitated DNA, we mapped genome-wide binding levels of KYP:3xFLAG. ChIP was performed using anti-FLAG antibody (sigma M2), and ChIP DNA were analyzed by Illumina NovoSeq.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of JMJ28 in 14 days old arabidopsis. By obtaining sequence from chromatin immunoprecipitated DNA, we generated genome-wide JMJ28-binding maps of 14 days old arabidopsis. To reveal bound genes by JMJ28, chimeric protein JMJ28-3xFLAG was expressed under JMJ28 promoter in jmj28 (JMJ28pro:JMJ28:3xFLAG/ jmj28). ChIP was performed using anti-FLAG antibody (FLAG-M2, F1804; SIGMA), and ChIP DNA were analyzed by Illumina Novaseq 6000.
Project description:ChIP-seq of Mycobacterium tuberculosis strain with a C-terminal FLAG-tagged WhiA allele compared to a strain carrying a FLAG attached to no protein (empty)
Project description:Histone modifications plays vital role in the regulation of immune response following Mycobacterium tuberculosis infection. Here, mouse macrophages (RAW264.7) were infected with M. tuberculosis (H37Rv) or left uninfected, followed by Chromatin immunoprecipitation with anti-H3K4me3 or anti-H3K27ac antibody and high throughput sequencing.
Project description:Approximately 20% of Acute Myelogenous Leukemia (AML) cases carry the t(8;21) translocation, which involves the AML1 and ETO genes, and express the resulting AML1/ETO fusion protein that functions as a transcriptional repressor by recruiting NCoR/SMRT/HDAC complexes to DNA. We used ChIP-chip to identify the determinants of AML1/ETO binding on a contiguous DNA region (chromosome 19). AML1/ETO binding regions are characterized by a specific sequence signature that includes the presence of the consensus binding sites for the AML1 and HEB transcription factors. We therefore assessed the binding patterns of AML1 and HEB on chromosome 19. A specific chromatin modification (tri-methylation of lysine 4 on histone 3 = 3MetK4) was also studied in U937 cells expressing AML1/ETO in order to correlate the identified binding profiles with active transcription sites. Keywords: ChIP-chip A U937 cell line that conditionally expresses HA-tagged AML1/ETO under the control of the mouse metallothionine promoter (U937-A1E) (Alcalay et al., J.Clin.Invest, 2003,112, 1751-1761) was used. Cell lines were treated for 8h with 100uM ZnSO4 to induce transgene expression in U937-A1E. We performed ChIP using anti-HA, anti-ETO, anti-AML1/RUNX1, anti-HEBor anti-3MetK4 antibodies. ChIP products were then PCR amplified, labeled with Cy3/Cy5 fluorescent dyes and hybridized to the NimbleGen custom made NGS_HG17_Chr.19Array. U937-Mt cells, which carry the empty vector, served as control (C) for non-specific antibody binding. Each sample identifier indicates Antibody_Cell line (example: HA_A1E = ChIP using anti HA antibody in U937-A1E cells; HA_C = ChIP using anti HA antibody in U937-Mt control cells)
Project description:ChIP-seq of mycobacterium tuberculosis expressing a 3xFLAG-tagged N-terminal WhiB2 compared with a strain carrying an empty FLAG (not attached to a protein)
Project description:we performed ChIP-seq assays to identify in vivo targets of GltR. The plasmid mini-gltR-flag-lacz was constructed and the resultant plasmid was fused to P. aeruginosa strain PAO1, yielding PAO1/mini-gltR-flag-lacz. We investigated GltR-binding to the chromosome of PAO1 during growth with glucose by ChIP-Seq. Sequence reads obtained from three independent ChIP-Seq experiments using anti-flag antibody and mapped to the P. aeruginosa PAO1 genome.Using the MACS software,we identified 55 enriched loci (q-value < 0.05) harboring GltR-binding peaks, that were enriched > 3-fold, but were absent in control sample conducted without anti-flag antibody.