Introduction of Bacterial Effector Proteins into Mammalian Cells by Electroporation
Trascrizione
Certain gram-negative bacterial pathogens contact host cells and employ secretion systems to inject effector proteins, facilitating cell invasion.
To deliver effector proteins into mammalian cells in vitro, pipette mammalian cells into a chilled cuvette. Add streptavidin-binding peptide-tagged bacterial effector proteins and mix.
Perform electroporation. During the run, short electric pulses disrupt the host cell membrane lipid bilayer to form transient pores, facilitating the cellular entry of effector proteins. Plate the cells onto a glass plate containing media, allowing the cells to recover and adhere to the plate. The pores reseal, entrapping the proteins within.
Following cell recovery, fix and permeabilize the cells for antibody access to intracellular targets. Incubate with a protein-containing blocking solution to prevent non-specific antibody binding. Add primary antibodies that specifically bind to the streptavidin-binding peptide tag on the intracellular effector proteins.
Introduce fluorophore-tagged secondary antibodies that bind to primary antibodies bound to effector proteins for visualization. Add fluorophore-conjugated wheat germ agglutinin and DAPI to stain cell membrane glycoproteins and DNA, respectively.
Using a confocal microscope, visualize fluorescence signals from effector proteins, cell membranes, and DNA.
Successfully electroporated cells exhibit significant intracellular fluorescent signals from effector proteins, suggesting their intracellular localization.
