Verification of internal binding of PDZ-domain and non-consensus C-terminally truncated protein partner by co-immunoprecipitation. (A) Schematic representation of both tagged proteins: PDZ domains carrying an N-terminal 3XHA epitope tag and C-terminally truncated (dCter) Y2H interacting protein fragment (MIR: experimentally defined minimal interaction region) carrying N-terminal MYC epitope. (B) All pairs were co-expressed in 293T cells and co-IPed using anti-HA sepharose beads. Binding of given protein upon precipitation was revealed by western blotting using anti-MYC serum. For each IP performed three panels are presented. Upper panel: IP reaction probed after resolution on SDS-PAGE and blotting with anti-HA antibody. Middle panel: the same IP reaction probed with anti-MYC serum detecting the truncated protein fragments (MYC:MIRdCter). Lower panel: detection of expression of each truncated protein fragment by probing total crude cellular extracts (input) with anti-MYC serum. The table identifies interaction pairs by their lane number and order in which they are presented in blot panels. The color code summarizes the outcome for each pair (purple: interaction tested positive, grey: no interaction detected and yellow: inconclusive as one or both partners were not expressed). Each MYC:MIRdCter construct used in above co-IP experiment was also subjected to co-transfection and co-immunoprecipitation with empty pDEST-CMV-3xHA vector to serve as a negative control for the binding assay (see Additional file 7).
Lenfant et al. BMC Genomics 2010 11:671 doi:10.1186/1471-2164-11-671