T

Reporter gene

Recognition site

FIGURE 26.21 Principle of Co-lmmunoprecipitation

To determine how many other proteins bind to a target protein, they are isolated by precipitating them together using an antibody. An antibody specific to the target protein is needed. If no specific antibody is available a widely used tag (such as FLAG) is added to the coding sequence. The target protein (orange) is cloned behind a mammalian promoter and expressed in mammalian cells where it will bind some other proteins (red, green, purple). The cytoplasmic fraction is isolated. Antibody to the target protein is added and the complexes are isolated by binding the antibody to beads coated with protein A. The beads are spun down. The components are then separated by SDS-PAGE to identify the number and size of the other proteins that bound to the protein of interest.

Mammalian promoter

Flag tag

Transfect INTO

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