Encyclopedia of Experiments: Biological Techniques
Ein Abonnement für JoVE ist erforderlich, um diesen Inhalt ansehen zu können. Melden Sie sich an oder starten Sie Ihre kostenlose Testversion.
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.