Effector Translocation Assay: Differential Solubilization

The identification of effector proteins delivered into mammalian host cells by bacterial pathogens possessing syringe-like nanomachines is an important step towards an understanding of the mechanisms underlying virulence of these pathogens. In this chapter, we describe a method based on mammalian tissue culture infection models where incubation with a non-ionic detergent (Triton X-100) enables solubilization of host cell membranes but not of bacterial membranes. This allows the isolation of a Triton-soluble fraction lacking bacteria but enriched in proteins present in the host cell cytoplasm, nucleus, and plasma membrane. Using appropriate controls, this fraction can be probed by immunoblotting for the presence of bacterial effector proteins delivered into host cells.