ABSTRACT: YFP-NPH3 was immunoprecipitated with GFP-Trap-A agarose beads from transgenic Arabidopsis plants, and then phosphorylation sites of NPH3 were identified.
Project description:Wild-type or S2265A version of GFP-RIF1 protein (isoform 1) was over-expressed in Flp-In-T-REx cells (Watts et al. 2020. eLife 9:e58020) and immunopurified using GFP-Trap magnetic agarose beads. Proteins were subjected to on-beads trypsin digestion and resulting peptides were analysed by mass spectrometry for protein identification/quantification and identification of phosphorylated residues. Raw MS file names and descriptions: IP_RIF1_A.raw = GFP-RIF1-L immunoprecipitated from cells without aphidicolin treatment. IP_RIF1_B.raw = GFP-RIF1-L immunoprecipitated from cells treated with 1 µM aphidicolin for 24 hr before harvesting. IP_RIF1_C.raw = GFP-RIF1-L (S2265A) immunoprecipitated from cells treated with 1 µM aphidicolin for 24 hr before harvesting.
Project description:We performed chloroplast ChIP-seq (cpChIP-seq) to identify the possible DNA-binding sites of mTERF5 in Arabidopsis thaliana. To this end, we generated transgenic Arabidopsis plants expressing mTERF5 carrying an HA tag under the control of the CaMV 35S promoter. Then, We used the polyclonal antibody (abcam, ab9110, lot GR304617-8 ) against HA tag which conjugated to ChIP-Grade protein A/G agarose (Thermo scientific, 26161, lot QJ223903) to perform cpChIP assay. The obtained chromatin immunoprecipitated DNA of chloroplasts were used to build DNA libaries for high-throughput sequencing. Finally, we showed that three potenssial DNA regions across the chloroplast genome compared to the control group were enriched by mTERF5.
Project description:To analyze phosphorylation sites of ALLO-1, worm eggs expressing GFP-ALLO-1 in the atg-11 or ikke-1 background were lysed in co-IP buffer, and GFP-ALLO-1 was immunoprecipitated with GFP-Trap-agarose beads. Proteins on the beads were directly digested with Trypsin overnight. Supernatants were collected, acidified to pH2.5 and desalted using GL-Tip SDB followed by PRM analysis.
Project description:To analyze phosphorylation sites of ALLO-1, worm eggs expressing GFP-ALLO-1 in the atg-11 or ikke-1 background were lysed in co-IP buffer, and GFP-ALLO-1 was immunoprecipitated with GFP-Trap-agarose beads. Proteins on the beads were directly digested with Trypsin overnight. Supernatants were collected, acidified to pH2.5 and desalted using GL-Tip SDB followed by LC-MS/MS analysis.
Project description:We profiled the gene expression patterns of undisturbed endothelial cells in living animals using a novel ‘AngioTag’ zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment. This transgenic line uses the endothelial cell-specific kdrl promoter to drive expression of an epitope tagged Rpl10a 60S ribosomal subunit protein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs. We also collected the whole embryo translatome using the TRAP protocol and a ubiquitously expressed tagged Rpl10a, and the endothelial transcritome by collecting the GFP+ endothelial cells that had the kdrl promoter driving GFP.
Project description:1-week-old seedlings of HDP4-YFP/hdp4-1 and GFP/col-WT transgenic plants under normal growth conditions were used for IP analysis. The GFP-Trap beads containing immunoprecipitated HDP4-YFP or GFP were washed twice with 1× PBS and dissolved in lysis buffer with 8M urea, 5 mM dithiothreitol and incubated at 50 °C for 30 min. Proteins were alkylated in 15 mM iodoacetamide for 1 h in the dark at 25 °C and then digested with trypsin (1:50) overnight at 37 °C. After digestion, chromatographic separation was performed using an Easy nLC 1200 chromatographic system (Thermo Fisher). Then, the peptides were analysed with the targeted parallel reaction monitoring method using a Velos LTQ-Orbitrap mass spectrometer (Thermo Fisher). The raw files were searched directly against Arabidopsis thaliana database (53,270 entries; UniprotKB) with no redundant entries using SEQUEST algorithm on Proteome Discoverer (Version 1.4; Thermo Fisher).
Project description:To understand the transcriptional effect of fasting and feeding a ketogenic diet on mouse CNS astrocytes, we performed translating ribosomal affinity purification (TRAP) of mRNAs immunoprecipitated from hippocampus. TRAP mice express a ribosomal epitope tag upon Cre-induced recombination that can be immunoprecipitated following activation. We measured the abundance of actively translating mRNAs from a ribosomal pull-down that came from adult astrocyte (Aldh1l1-Cre)-specific TRAP mice that were subjected to one of three dietary conditions: four weeks of normal chow diet, four weeks of ketogenic diet (high-fat, low-carbohydrate)43, or an 18-hour fast. Immediately following the respective diets, forebrain and hippocampus was harvested from all groups, ribosomes were immunoprecipitated, and actively translating mRNAs in the ribosomes were purified.
Project description:This project aims at identifying the binding partners of the Arabidopsis thaliana MRF7 protein and of its truncated form ΔMRF7, which lacks a potential protein binding domain DUF593 at the N-terminus. A.thaliana plants stably overexpressing GFP-MRF7 and GFP-ΔMRF7 were generated by floral dipping of Col-0 plants. Co-immunoprecipitation was carried out using the GFP-Trap®_A beads technology (Chromotek) and samples thus obtained were analysed through Liquid Chromatography-Mass Spectrometry
Project description:To analyze proteins copurified with GFP-ALLO-1, worm eggs expressing GFP-ALLO-1 or GFP in the atg-11 background were lysed in co-IP buffer, and GFP-ALLO-1 or GFP was immunoprecipitated with GFP-Trap-agarose beads. Proteins on the beads were directly digested with Trypsin overnight. Supernatants were collected, acidified to pH2.5 and desalted using GL-Tip SDB followed by LC-MS/MS analysis.
Project description:Myc-tagged N-terminal portion of TOC1 or TOC1 mutant (S194A, S201A, T204A) were expressed in tobacco leaves and then immunoprecipitated. The immunoprecipitated samples were separated on SDS-PAGE gels, and then phosphorylation sites of TOC1 were identified from gel bands.