Encyclopedia of Experiments: Biological Techniques
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Transcript
In cells, proteins bind to their interacting proteins via specific sequences in their structure ― modulating intracellular protein functioning.
To detect intracellular protein interactions, take an adherent cell culture. Aspirate the medium.
Add a biotinylated cell-penetrating peptide, BCPP solution, and incubate.
Each BCPP contains a peptide sequence that helps in specific binding to the targeted intracellular protein. The peptide has a cell-penetrating peptide, CPP, at the peptide's N-terminus and biotin at the C-terminus for easy detection.
During incubation, the positively-charged CPP region facilitates bound peptide entry through the cell's membrane, reaching the cytoplasm. The peptide binds to its intracellular interacting protein partner ― forming a BCPP-target protein complex.
Add a buffer containing detergent molecules that rupture the cell's membrane and release BCPP-target protein complexes and other cellular components.
Add a solution of agarose bead-bound avidin — a glycoprotein that specifically binds to the biotin in BCPPs. Incubate, allowing the irreversible binding of avidin to biotin and purifying avidin-BCPP-target protein complexes.
Centrifuge to pellet the beads. Resuspend the beads in a buffer to cleave non-covalent linkages between the proteins and elute CPP-peptide-protein complexes from the biotin-avidin beads.
Analyze the CPP-peptide-protein complexes by western blotting. A higher molecular weight band for the CPP-peptide-protein complex than the complex's components indicates a successful interaction between the interacting protein partners.
