Project description:Immunoprecipitation of endogenous Myosin VI in Human Cell Lines (Caco-2, HeLa, MCF10A, MDA-MB-231) and identification of true interactors by mass spectrometry
Project description:To characterize the cellular targets of the ASFV pI73R, we carried out an Immunoprecipitation Mass-Spectrometry (IP-MS) experiment. Whole-cell extracts prepared from PAM cells which infected with ASFV I73R-mHA were used to immunoprecipitate endogenous targets pI73R, with a matched IgG serving as a control. The immunoprecipitated proteins were then subjected to LC-MS/MS.
Project description:We have measured absolute copy numbers and dynamics of cohesin, CTCF and their regulators NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells before and after DNA replication. Comparison of these numbers with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time.
Project description:Co-immunoprecipitation of recombinantly expressed IPO9 using a strep-tag in HeLa cell lysate to identify IPO9 interaction partners.
Project description:To investigate the functions of REIIBP, we conducted the immunoprecipitation (IP)-mass spectrometry analysis to identify the interacting proteins of REIIBP.
Project description:The project was aimed at identifying binding partners of endogenous SLX4 in HeLa cells. Whole cell extracts from HeLa Flp-In T-Rex cells were used for immunoprecipitation performed with anti-SLX4 antibodies from Bethyl Laboratories (A302-270A and A302-269A), either used alone or in combination. To discriminate bona fide SLX4 partners from non-specific proteins recognized by the anti-SLX4 antibodies, cells were transfected with control (Luc) siRNA or a siRNA targeting SLX4. After immunoprecipitation, protein complexes were analysed by LC-MSMS.
Project description:Endogenous condensates with transient constituents are notoriously difficult to study with common biological assays like mass-spectrometry and other proteomics profiling. Here we report a method for light-induced targeting of endogenous condensates (LiTEC) in living cells. LiTEC combines the identification of molecular zip codes that target the endogenous condensates with optogenetics to enable controlled and reversible partitioning of an arbitrary cargo, such as enzymes commonly used in proteomics, into the condensate in a blue light dependent manner. We demonstrate a proof of concept by combining LiTEC with proximity-based biotinylation (BioID) and uncover putative components of transcriptional condensates in mouse embryonic stem cells. Our approach opens the road to genome-wide functional studies of endogenous condensates.