Project description:We applied 4-thiouridine to cultured cells expressing the FLAG/HA-tagged RNA-binding proteins (RBPs) followed by UV 365 nm irradiation. The crosslinked RNA-protein complexes were isolated by immunoprecipitation, and the covalently bound RNA was partially digested with RNase T1 and radiolabeled. The radiolabeled RNPs were subsequenctly separated by SDS-PAGE, the crosslinked RNA segments recovered and converted into a cDNA library and sequenced.
Project description:To identify the target of the monoclonal antibody, Immuoprecipitation (IP) was performed to pull-down target protein from cell culture extraction, corn or zebrafish tissue lysate. The IP product was validated on silver-stained SDS-PAGE and Western blot. The target band was cut out and performed LC-MS/MS for target protein identification.
Project description:Identification of targets of the protein disulfide reductase thioredoxin using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and thiol specific differential labeling with isotope-coded affinity tags (ICAT). Reduction of specific target disulfides is quantified by measuring ratios of cysteine residues labeled with the heavy (13C) and light (12C) ICAT reagents in peptides derived from tryptic digests of Trx-treated and non-treated samples. Keywords: protein, LC-MS/MS, ICAT
Project description:We applied 4-thiouridine to cultured cells expressing the FLAG/HA-tagged RNA-binding proteins (RBPs) followed by UV 365 nm irradiation. The crosslinked RNA-protein complexes were isolated by immunoprecipitation, and the covalently bound RNA was partially digested with RNase T1 and radiolabeled. The radiolabeled RNPs were subsequenctly separated by SDS-PAGE, the crosslinked RNA segments recovered and converted into a cDNA library and sequenced. RBPs were UV-crosslinked to their RNA targets containing 4-thiouridine. The RNA segments were recovered after immunoprecipitation and seqeunced by Solexa.
Project description:We evaluated the effect of the different solubilization techniques on the reproducibility and width of the chorionic proteome characterized using bottom-up proteomics: SDS-based solubilization coupled with in-gel protein digestion after applying self-named 1DE gel-concentration procedure (SDS-PAGE without fractionating in the resolving gel for SDS removal) and in-solution protein digestion of urea-thiourea extracts. These results indicated that 1DE-concentration procedure coupled with in-gel digestion, LC-MS/MS and combinational usage of different bioinformatics tools provides in-depth analysis of a single protein band, including reliable identification and quantification of low-abundant proteins, e.g. FUCA1, PSG7.
Project description:This study aims to investigate lysine acetylation sites on the H-NS protein in Edwardsiella piscicida. The H-NS protein was first purified, then subjected to SDS-PAGE, followed by gel band excision and LC-MS/MS identification.