[{"id":467,"title":"Cros-IP: A Novel Co-immunoprecipitation Method for Characterising Direct/Indirect Protein Interactions.","sha":"22350c3ea4cda69378dd740bd47ad8a3","user_id":null,"state":"published","body":"Co-immunoprecipitation (co-IP) is considered the gold standard in vitro technique for characterising protein-protein interactions. However, the technique is limited by the inability to differentiate direct/indirect interactions of proteins. This publication presents a novel IP technique named ‘Cros-IP’ (Figure 1: Protocol Flow Diagram) that has been validated by experiments (data not shown). Antibody immobilised to beads containing Protein A/G is incubated with cell lysate/protein mixture, and spun/washed using the conventional co-IP method, with the exception that the tris-based buffers should be replaced by phosphate-based buffers due to the presence of a primary amine on the tris compound. After unbound (non-interacting) proteins in the cell lysate have been thoroughly washed away, the beads-antibody-protein complex is incubated with the protein crosslinker disuccinimidyl suberate (DSS) for 2 hours on ice. The protein complex is then eluted using a sodium dodecyl sulfate (SDS)-based buffer. This technique takes advantage of the considerably inconsistent crosslinking rate of DSS, with a series of crosslinked protein products expected to be formed. The product consisting the antigen (target protein) crosslinked with interacting proteins (Figure 1, Product B) can be used to characterise the sequence of protein interactions of an in vivo protein complex via SDS-PAGE/Western blotting and/or mass spectrometry characterisation.","created_at":"2018-06-26T23:46:54.590Z","updated_at":"2018-06-27T06:01:26.467Z","zenodo_id":1298748,"doi":"https://doi.org/10.5281/zenodo.1298748","subject":null,"tags":["biochemestry","co-immunoprecipitation","mass spectrometry","proteomics","protein","crosslink"],"vote_count":0,"view_count":1,"score":0.1,"deleted":false,"muted":false,"tweeted":true,"attachment_file_name":"Figure1.jpg","attachment_content_type":"image/jpeg","attachment_file_size":386880,"attachment_updated_at":"2018-06-26T23:46:54.379Z"}]