Biotinylated Cell-Penetrating Peptide Probe to Detect Protein-Protein Interactions

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.