NGFN-PLUS

Proteomic mapping of interactors from in vivo and in vitro systems

Coordinator:    Prof. Dr. Matthias Mann
Institution: Max-Planck-Institute für Biochemie, Martinsried
Homepage: www.biochem.mpg.de/mann/
In this subproject we will use the power of proteomics to obtain functional information of the genes of interest to the IG. Specifically, we will determine interaction partners using quantitative proteomics in the form of Stable Isotope Labeling by Amino acids in Cell culture (SILAC), to identify true interactors including both sub-stoichiometric and transient ones in a vast excess of background binders. The technology will be used on immunoprecipitates of tagged proteins expressed from BACs and/or endogenous loci to secure physiological expression levels rather than the transiently overexpressed proteins usually used in proteomic interaction screens, thereby removing a large obstacle in the search for relevant in-vivo binding. The technology will be used on both mouse and human cells, including stem cells differentiated to neuronal precursors. We will also employ high resolution mass spectrometry to quantify the whole proteomes of selected knock-out or knock-down cell lines, yielding both destabilized direct interaction partners and a general functional profile of the genes of interest.


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