SPOmics-Interactome is the platform for the study of protein-protein interactions at IPS2. The technologies it masters are based on the only method allowing the detection of these interactions at high throughput and in vivo - the yeast two-hybrid system (Y2H) - today optimized and automated. Thanks to the use of 384-well plates and a robotized system on the one hand, but also to a protocol entirely in liquid medium, the platform is able to screen a pool of 50 hybrid proteins (DB- X) against the AD-AIM hybrid protein library (about 12000 Arabidopsis proteins) in two months.
The protocol generally used is as follows: Plasmids carrying the ORFs encoding the proteins of interest provided by our collaborators in the pDEST - DB vector are used to transform yeast, then the hybrid DB-X proteins are tested to eliminate those capable of self-activation. Yeast expressing the hybrid DB-X proteins are then grown individually in selective media, combined into pools of 50 clones and screened against the AD-AIM library. After identification of candidate proteins, Y2H matrix analysis is performed by "deconvolution" of the 50 DB-proies and individual testing against the AD-bait candidates. A final sequencing step of the AD- and DB- proteins allows validation of the identity of the interacting proteins. Each new ORF screened is integrated into the AD- library allowing a constant implementation of the network.
Lab of attachment
IPS2, plateau du Moulon (bât 630)