Tissue- and isoform-specific protein complex analysis with natively processed bait proteins

ABSTRACT: Protein-protein interaction analysis is an important tool to elucidate the function of proteins and protein complexes as well as their dynamic behavior. To date, the analysis of tissue- or even cell- or compartment-specific protein interactions is still relying on the availability of specific antibodies suited for immunoprecipitation. Here, we aimed at establishing a method that allows identification of protein interactions and complexes from intact tissues independent of specific, high affinity antibodies used for protein pull-down and isolation. Tagged bait proteins were expressed in human HEK293T cells and residual interactors removed by SDS. The resulting tag-fusion protein was then used as bait to pull proteins from tissue samples. Tissue-specific interactions were reproducibly identified from porcine retina as well as from retinal pigment epithelium using the ciliary protein lebercilin as bait. Further, murine heart-specific interactors of two gene products of the 3’,5’-cyclic guanosine-3’,5’-monophosphate (cGMP)-dependent protein kinase type I were investigated. Here, specific interactions were associated with the Iα and Iβ gene products, that differ only in their unique amino-terminal region comprising about 100 AS. As such, the new protocol provides a fast and reliable method for tissue-specific protein complex analysis which is independent of the availability or suitability of antibodies for immunoprecipitation.

INSTRUMENT(S):

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

SUBMITTER: Tina Beyer  

LAB HEAD: Marius Ueffing

PROVIDER: PXD020379 | Pride | 2023-12-28

REPOSITORIES: Pride