EpiGex has developed a powerful method called EpiDiscoverTM for a fast, sensitive and accurate analysis of protein-protein interaction networks using Triple affinity Purification (TAP) of native complexes and mass spectrometry analysis. The TAP System allows you to recover interacting proteins from mammalian cells. The method is based on expression of a protein of interest fused to three affinity tags: a FLAG peptide, a HA peptide and a 6 His peptide. Triple affinity purification yields your tagged protein and interacting partners using gentle washing and small molecule elution conditions. There is no need for protease digestion to recover interacting protein partners since FLAG, HA and His elute easily from their affinity resins, leaving your interacting proteins intact and contaminant free. Aliquots of the purified protein preparation are subjected to a gel-free trypsin digestion and directly analyzed using LC- MS/MS. The purified complexes are also useful for downstream applications like enzymatic activity assays.
The use of EpiDiscoverTM allows a much faster, cheaper and more accurate method for dissecting protein-protein interaction networks:
- Clear identification of the partners involved in the complex.
- Specific: only the specific complexes are detected.
- Sensitive: Only a few hundred femtomol of protein samples are required to achieve the analysis.
- Stoechiometry of the complex: unique information for understanding the protein composition of the complex and the Protein-Protein Interaction network.
Three-Step Purification Protocol
Purifying proteins with the TAP protocol requires three steps (Figure 1). Since each purification step provides a different contaminating background, the combined protocol yields exceptionally clean proteins. For the first purification step, the mammalian cell lysate is applied to an anti-FLAG column; elution is challenged by using FLAG peptide. Next, the eluate is applied to an anti-HA column for the second purification step; elution is induced using HA peptide. Finally, the eluate is applied to a Nickel column for the third purification step; and the protein of interest with its binding partners is released by imidazole competition. Because our elution conditions are extremely gentle, the interacting proteins are not disrupted, and you may further analyze the proteins using routine techniques like Western blotting, mass spectrometry and enzymatic assays.
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