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#TAP

2 public questions tagged with this topic.

Protein-Protein Interaction

Which protease is used in TAP?

Tandem Affinity Purification requires a highly specific, mild protease to release first affinity complex without disrupting interacting partners. Tobacco Etch Virus protease recognizes seven amino acid sequence ENLYFQG and cleaves between Q and G with high specificity under low temperature, physiological pH, and in presence of mild detergents that preserve protein-protein interactions. This orthogonal cleavage site is engineered between Protein A and CBP modules. Trypsin, caspase, and collagenase are more promiscuous or require conditions that would dissociate or degrade complexes, unsuitable for preserving interactome.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Protein-Protein Interaction

The first step in TAP is:

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.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details