Project description:Binding partners of CERS6 were identified by co-immunoprecipitation, and a liquid chromatograph and a tandem mass spectrometry analysis
Project description:To identify immediate early signaling components in the FLS2 pathway we performed co-immunoprecipitation (co-IP) experiments with FLS2-GFP as bait in Arabidopsis. We immunoprecipitated FLS2-GFP with anti-GFP antibody coated beads and analyzed co-precipitated proteins by liquid chromatography tandem mass spectrometry (LC-MS/MS).
Project description:To investigate whether GltB possess more function, Co-immunoprecipitation coupled with Mass Spectrometry (CoIP-MS) was used to isolate the putative GltB binding proteins.We used strains PAO1/p-gtrS-YFP and ΔgltB/p-gtrS-YFP to ensure the membrane protein GtrS of great interest is at high levels. A target GltB-specific antibody anti-GltB was used and ΔgltB/p-gtrS-YFP was set up as a negative control. The whole gel lane was divided into small fractions and in-gel digested for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Control sample was also analyzed in parallel to distinguish the background proteins.
Project description:Results from co-immunoprecipitation (co-IP)–MS analysis of VIPP-YFP bait, expressed in wild-type Arabidopsis thaliana. GFP-Trap magnetic agarose beads were used for co-IP. Co-precipitated peptides were digested on the beads and analyzed by liquid chromatography–mass spectrometry (LC-MS). The MS data were processed using the Philosopher toolkit, with MSFragger as the search engine, followed by IonQuant. SAINTexpress software was used to score potential interactions between the observed proteins (potential prey) and the bait protein. It is recommended to use .raw files for reanalysis.
Project description:Results from co-immunoprecipitation (co-IP)-MS analysis of VIPP-YFP bait, expressed in Arabidopsis thaliana via1 mutant line. GFP-Trap magnetic agarose beads were used for co-IP. Co-precipitated peptides were digested on the beads and analyzed by liquid chromatography-mass spectrometry (LC-MS). The MS data were processed using the Philosopher toolkit, with MSFragger as the search engine, followed by IonQuant. SAINTexpress software was used to score potential interactions between the observed proteins (potential prey) and the bait protein. It is recommended to use .raw files for reanalysis.
Project description:The goal of this study is to compare the mRNA interactome of different RBPs in regenerating axons utilizing RNA-immunoprecipitation (RIP). Interacting mRNAs to each target RBPs were co-immunoprecipitated from axoplasm of sciatic nerve, injured 7 days ago. Interactome of each RBPs were identified by Next-generation sequencing (NGS).
Project description:Identification of CHKA-binding proteins under glutamine deprivation by co-immunoprecipitation (Co-IP) coupled with mass spectrometry (MS)
Project description:293T cells were transfected with pCMV CEBPbeta LAP2 isoform. Two days post transfection, nuclear extracts were subjected to Co immunoprecipitation with mouse anti-CEBPbeta (clone H-7) or mouse control IgG using Protein G Dynabeads. After washing with RIPA buffer, beads were subjected to Mass spectrometry based protein identification to find interacting partners of CEBPbeta.
Project description:CHD (chromodomain helicase DNA binding protein) family is composed of nine members of chromatin remodeling factors that regulate chromatin structure in an ATP-dependent manner. Among them, CHD4 contributes to many basic cellular functions during development mainly through multiple proteins interacting with CHD4 including NuRD (nucleosome remodeling and deacetylase activities) complex, which contains histone deacetylase HDAC1/2. However, functions of CHD4 that are not mediated by NuRD complexes have also been found, implying the existence of unknown proteins that are associated with CHD4. In the present study, we generated CHD4 FLAG-tag knock-in cells in HeLa-S3 and HEK293T cells and sought proteins bound to CHD4 with the use of immunoprecipitation and liquid chromatography and tandem MS (LC-MS/MS) analysis. We found that LCORL (ligand dependent nuclear receptor corepressor like) and NOL4L (nucleolar protein 4 like) were reproducibly identified as a novel CHD4 interactors. RNA-sequencing analysis of HEK293T cells depleted of CHD4, LCORL, and NOL4L revealed consistent upregulation of genes related to the Notch pathway. Our results thus suggest that both NOL4L and LCORL may cooperate with CHD4 to suppress the Notch pathway in mammalian cells.
Project description:Shotgun LC-MS/MS was performed on immunoaffinity purified TBC1D15-interacting proteins. We searched for in vivo partners of TBC1D15-mediated regulation by large-scale immunoaffinity purification of interacting proteins of endogenous TBC1D15 in TICs and Identification of interaction partners was accomplished by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach identified several high-confidence interacting proteins, including NuMA1, NOTCH1/2/3/4 and RANGAP1.