Add Beads Coated With Protein A

Other proteins

Protein complexes

1. SPIN OUT bead COMPLEXES 2. RUN on sDS-PAGE

3 associated proteins

FLAG-tagged protein

FIGURE 26.22 Confirmation of Protein Interaction using Co-Immunoprecipitation

To determine if protein X and Y interact in cultured mammalian cells, they must be fused to two different tag sequences (His6 tag and FLAG tag in this example). The two expression plasmids are transfected into the same mammalian cells. The cytoplasmic protein fraction is isolated and divided into two samples. Antibody to the FLAG tag is added to one sample and antibody to the His6 tag is added to the other. The antibody/protein complexes are isolated using Protein A beads. Each sample is then tested for the presence of the other protein by antibody directed against its tag. That is, the proteins precipitated along with protein X-FLAG are tested for the presence of protein Y. And conversely the proteins precipitated along with protein Y-His6 are tested for protein X.

Gene for protein X

Gene for protein Y

Gene for protein X

Gene for protein Y

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