Bimolecular Complementation Affinity Purification to Isolate Two Interacting Proteins

To detect and isolate a pair of specific interacting proteins using bimolecular complementation affinity purification, add a pair of bimolecular fluorescence complementation plasmids-transfection agent mixture into a culture plate containing mammalian cells.

One plasmid encodes one interacting protein — the bait protein — fused to a reporter fluorescent protein fragment. The other plasmid encodes for the other binding protein — the prey protein — fused to the fluorescent protein's complementary fragment.

Incubate. The transfection agent facilitates plasmid cellular uptake. Successfully transfected cells express cell membrane-localized proteins.

The bait-prey proteins' interactions bring the fluorescent protein fragments closer. This causes the fragments to fuse and refold, forming the functional fluorescent protein, whose fluorescence can be visualized under a fluorescence microscope.

Replace the media with a non-ionic detergent-containing lysis buffer to disrupt the cellular membranes, releasing the fusion proteins. Collect the cell lysate in a tube. Centrifuge.

Transfer the fusion protein-containing supernatant into a fresh tube. Add agarose beads conjugated with the fluorescent