Practice question

TAP workflow is designed for high purity through two orthogonal affinities. Lysate containing TAP-tagged bait first incubates with IgG sepharose, where Protein A domain binds heavy chain efficiently. Unbound proteins are washed extensively. Bound material is then released by TEV protease cleavage that separates Protein A from CBP-bait, ensuring specific elution. Second affinity over calmodulin beads in presence of calcium captures CBP. Calmodulin binding never occurs first, and mass spectrometry follows after final EGTA elution. This order minimizes contaminants before identification.