Tandem Affinity Purification Assay to Study Protein-Protein Interactions
Transcrição
Tandem affinity purification is a sequential purification scheme for isolating protein complexes from cells to study protein-protein interactions.
To begin, take a cell culture expressing epitope-tagged proteins of interest. The proteins form a complex inside the cell. One of the proteins in the complex contains a Strep-tag, while the other is labeled with a FLAG-tag. These are small synthetic peptide sequence-based affinity tags.
Remove the medium, and add a detergent solution to lyse the cells. Centrifuge to remove the cellular debris. Collect the supernatant containing the target protein complex and other cellular proteins.
Add Strep-beads — agarose beads coated with streptavidin — and incubate. The Strep-tag of the protein complex binds to streptavidin. Centrifuge to collect the protein complex-bound beads. Wash to remove any contaminating cellular proteins.
Add elution buffer containing desthiobiotin — a biotin derivative — which displaces the protein complex by competitively binding to streptavidin. Centrifuge to obtain a supernatant containing the eluted protein complex.
Add FLAG-beads — agarose beads coated with anti-FLAG antibodies — and incubate. The FLAG-tag of the protein complex binds to the antibody. Centrifuge to collect the protein complex-bound beads.
Add elution buffer containing free FLAG peptide. The peptide competitively binds to the antibody and displaces the bound protein complex. Centrifuge to elute the purified protein complex.
Assess the presence of both proteins in the elute to confirm the interaction between the proteins of interest.
