Tandem Affinity Purification Assay to Study Protein-Protein Interactions

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 prote