Encyclopedia of Experiments: Biological Techniques
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In its active form, plasma membrane-associated Ras monomer ― a guanine nucleotide-binding protein ― recruits Raf, an inactive protein kinase that binds to Ras via its Ras-binding domain.
This binding results in a functional Ras-Raf complex that triggers the downstream signaling process.
To visualize the intracellular Ras-Raf interaction, begin with a suspension of genetically-engineered cells in a well-chambered slide. These cells express two non-fluorescent fusion proteins ― one comprises Ras linked to one-half of a photoactivatable fluorophore via its N-terminal, and the other is Raf fused to the other half of the same fluorophore at its C-terminal.
Inside cells, Ras-containing fusion proteins dock at the plasma membrane and interact with Raf-containing fusion proteins, reconstituting the fluorescent protein's fragments to a complete photoactivatable fluorophore. The absence of Ras and Raf binding keeps the two fluorophore halves separated.
Add a fixative reagent to the slide. The fixative molecules form cross-linkages with the proteins and stabilize the protein complexes.
Remove the fixative reagent and add an imaging buffer. Place the slide under a fluorescence microscope set for photoactivated localization microscopy, PALM. Illuminate the cells with a high-intensity laser, activating a small subset of fluorescent molecules.
Image the randomly-originating fluorescence produced by each nano-sized, single fluorophore-tagged Ras-Raf complex, visible as high-resolution fluorescence dots.