FILIP1 CT pull-down

FILIP1 was identified as potential new filamin C interaction partner by a SILAC-based BioID experiment. Interaction of the two protein via the carboxy-terminal end of FILIP and domain 18-21 of filamin C was prooven by yeast-2-hybrid sreens and dot-blot assays, respectively. To verify this interaction by mass spectrometry, recombinantly expressed FILIP1 carboxy-term was fused via its His6-tag to Ni2+-NTA beads. Subsequently. beads were incubated with cell extracts from human skeletal muscle cells. Bands observed in SDS-PAGE from the pull-down assay were cut and analysed by LC-MS. As a result Filamin C could be identified as FILIP1 binding partner.