Project description:Here we show that biotin-labelled miR-34a can be loaded to AGO2, and AGO2 immunoprecipitation can pulldown biotinylated miR-34a (Bio-miR pulldown). RNA-sequencing (RNA-seq) of the Bio-miR pulldown RNAs efficiently identified miR-34a mRNA targets, which could be verified with luciferase assays. In contrast to the approach of Bio-miR pulldown, RNA-seq of miR-34a overexpression samples had limited value in identifying direct targets of miR-34a. It seems that pulldown of 30 -Biotin-tagged miRNA can identify bona fide microRNA targets at least for miR34a.
Project description:CD34 positive hematopoietic stem cells were differentiated into erythroid lineage. Next generation sequencing (NGS) of 5hmC affinity pulldown and RNAseq were performed in four time point of different stages of erythroid differentiation. 4 RNA-Seq Samples (d0, d3, d7 and d10); 4 affinity-pulldown (d0, d3, d7 and d10), and 4 input samples (d0, d3, d7 and d10).
Project description:We performed pulldown assays followed by mass-spectrometry analysis using biotin-tagged 13-cis retinoic acid to identify its binding targets in HeLa cells
Project description:CD34 positive hematopoietic stem cells were differentiated into erythroid lineage. Next generation sequencing (NGS) of 5hmC affinity pulldown and RNAseq were performed in four time point of different stages of erythroid differentiation.
Project description:"Here, we report the development of a novel version of Kinobeads that extends kinome coverage to PIKK and PI3K kinases. This is achieved by inclusion of two affinity probes derived from the clinical PI3K/MTOR inhibitors Omipalisib and BGT226. The new affinity matrix was used to profile 13 clinical and pre-clinical PIKK/PI3K inhibitors. The large discrepancies between the PI3K affinity values obtained and reported results from recombinant assays led us to perform a phosphoproteomic experiment showing that the chemoproteomic assay is the better approximation of PI3K inhibitor action in-vivo."
Project description:Heme-affinity pull-down assays and proteomics of lysates from primary cortical neurons identified and EAG channel, hERG3 (Kv11.3) binds to heme.
Project description:Here we report an unexpected chemistry through which ITC-protein adduct forms a stable N-terminal dihydrothiazole peptide adduct during proteolysis. This proteolysis-assisted cyclization (PAC) reaction can be harnessed for developing affinity-based and activity-based chemoproteomic methods to site-specifically profile targets of ITCs.
Project description:We report the application of RNA-sequencing to streptavidin-affinity purified RNA transcripts from HeLa cell lysates. RNA was biotinylated using the enzymatic RNA-TAG methodology using two variants of E. Coli TGT, the wild type enzyme and an obligate dimeric form of the enzyme for comparison of selectivity. From this analysis, it was determined that the obligate dimer enabled affinity purification of the desired transcript with much greater selectivity.
Project description:Cysteine sulfinic acid or S-sulfinylation is an oxidative post-translational modification (OxiPTM) that is known to be involved in redox-dependent regulation of protein function but has been historically difficult to analyze biochemically. To facilitate the detection of S-sulfinylated proteins, we demonstrate that a clickable, electrophilic diazene probe (DiaAlk) enables capture and site-centric proteomic analysis of this OxiPTM. Using this workflow, we revealed a striking difference between sulfenic acid modification (S-sulfenylation) and the S-sulfinylation dynamic response to oxidative stress, which is indicative of different roles for these OxiPTMs in redox regulation. We also identified >55 heretofore-unknown protein substrates of the cysteine sulfinic acid reductase sulfiredoxin, extending its function well beyond those of 2-cysteine peroxiredoxins (2-Cys PRDX1-4) and offering new insights into the role of this unique oxidoreductase as a central mediator of reactive oxygen species-associated diseases, particularly cancer. DiaAlk therefore provides a novel tool to profile S-sulfinylated proteins and study their regulatory mechanisms in cells.