Project description:To identify Arabidopsis Wdr8 interactors, immunoprecipitation associated LC-MS/MS analysis was carried out. Crude proteins extracted from Wdr8-GFP expressing transgenic plants was subjected to the immuno-precipitation assay using GFP antibody magnetic beads (µMACS GFP isolation kit, Miltenyi Biotec). Co-purified proteins were separated by 10% SDS-PAGE gel and stained with SYPRO Ruby (BioRad laboratories). The stained protein bands were excised into several fraction pieces according to protein sizes, and in-gel protein digestion by trypsin was carried out. Purified protein peptides were subjected to LC-MS/MS analysis (LTQ-Orbitrap XL-HTC-PAL system). Collected MS/MS peak spectra was analyzed by the MASCOT server to identify peptide sequence.
Project description:Numerous lineage analysis studies have used FACS sorting as a separation technique prior to measuring mRNA expression patterns. To assess if FACS sorting causes non-specific changes in gene expression, we collected a heterogeneous population of mammary epithelial cells from three biological replicates. We then performed gene expression analysis on each replicate either prior to trypsin digestion, immediately following trypsin digestion, or following both the trypsin digestion and a mock FACS sort. In this dataset, we include gene expression data obtained from isolated murine mammary epithelial cells either prior to trypsin digestion, immediately following trypsin digestion, or following the trypsin digestion and a mock FACS sort.
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 studied the effect of Ser30 with or without O-GlcNAc modification on K18 protein and its co-immunoprecipitated proteins in cholangiocarcinoma cells.The lysates from HuCCT1 shK18 stable cell line transfected with FLAG-K18-WT or FLAG-K18-S30A were captured with anti-FLAG beads, and then subjected to in-gel trypsin digestion and LC-MS/MS analysis.
Project description:In an attempt to identify proteins interacting with transcription factor E2F2, proteins extracted from HA-E2F2 overexpressing HEK293T cells were were subjected to immunoprecipitation (IP) with an anti-HA antibody or, as a negative control (NegCt), with an anti-T antibody. Immunoprecipitated proteins were eluted, digested with trypsin, and the resulting peptides were analyzed by LC-MS/MS. The whole procedure was repeated several times in an attempt to gauge reproducibility (nine replicates of the anti-HA IP and six replicates of the control IP).
Project description:Triplicate samples of oocyte and cumulus cells were isolated from follicles of 2-8 mm, containing oocytes with several layers of cumulus cells were collected from ovaries. Proteins were extracted using DDF and digested using trypsin. The peptides were desalted, separated using in-line SCX-RPLC and analyzed using ion trap MS/MS. Proteins were identified using TurboSEQUEST 3.2
Project description:Numerous lineage analysis studies have used FACS sorting as a separation technique prior to measuring mRNA expression patterns. To assess if FACS sorting causes non-specific changes in gene expression, we collected a heterogeneous population of mammary epithelial cells from three biological replicates. We then performed gene expression analysis on each replicate either prior to trypsin digestion, immediately following trypsin digestion, or following both the trypsin digestion and a mock FACS sort. In this dataset, we include gene expression data obtained from isolated murine mammary epithelial cells either prior to trypsin digestion, immediately following trypsin digestion, or following the trypsin digestion and a mock FACS sort. Nine total samples were analyzed (3 biological replicates of three experimental conditions). Using LIMMA packages gene-wise comparisons were made between untrypsinized and trypsinized replicates as well as between trypsinized and mock FACS sorted replicates using and Pâ¤0.05 and a â¥1.5-fold difference between conditions.
Project description:HEK293T cells were ectopically expressing Flag tagged p62 (p62 group) or empty vectors (Control group) for 48h, cell lysates were incubated with anti-Flag affinity gels, and co-immunoprecipitates were subjected to trypsin digestion followed by mass spectrometry analysis. Peptides were separated by the EASY-nLC system (Thermo Fisher) and analyzed by the Q Exactive mass spectrometer (Thermo Fisher). Protein analysis were performed with Thermo Proteome Discoverer 2.1 (Thermo Fisher) and searched against Uniprot Human database. Three samples per group.
Project description:HEK293T cells were ectopically expressing Flag tagged p62 (p62 group) or empty vectors (Control group) for 48h, cell lysates were incubated with anti-Flag affinity gels, and co-immunoprecipitates were subjected to trypsin digestion followed by mass spectrometry analysis. Peptides were separated by the EASY-nLC system (Thermo Fisher) and analyzed by the Q Exactive mass spectrometer (Thermo Fisher). Protein analysis were performed with Thermo Proteome Discoverer 2.1 (Thermo Fisher) and searched against Uniprot Human database. Three samples per group.
Project description:LN229 cells were subjected to Co-IP using IgG and anti-ECE1 antibodies. The immunoprecipitated complexes were validated by Western blot and subsequently analyzed by liquid chromatography–mass spectrometry (HPLC-MS/MS, QLBio, China) for identification of ECE1-interacting proteins.