SILAC-iPAC: a quantitative method for distinguishing genuine from non-specific components of protein complexes by parallel affinity capture.

ABSTRACT: Pull-down assays can identify members of protein complexes but suffer from co-isolation of contaminants. The problem is particularly acute when the specifically interacting partners are of low-abundance and/or bind transiently with low affinity. To differentiate true interacting partners from contaminants, we have combined SILAC labelling with a proteomic method called "Interactomes by Parallel Affinity Capture" (iPAC). In our method, a cell-line stably expressing a doubly tagged target endogenous protein and its tag-less control cell-line are differentially SILAC labelled. Lysates from the two cell-lines are mixed and the tagged protein is independently purified for MS analysis using multiple affinity resins in parallel. This allows the quantitative identification of tagged proteins and t

SUBMITTER: Rees JS 

PROVIDER: S-EPMC4329988 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature