Affinity purification, immunoprecipitation, and mass spectrometry analysis are essential tools used in proteomic studies. A successful outcome is facilitated by high-capacity supports, efficient sample preparation, and reproducibility provided by automation. We demonstrate that an anti-DYKDDDDK magnetic agarose fulfills these criteria. The DYKDDDDK epitope tag is commonly used in protein expression systems as a short purification handle. Using an antibody against a DYKDDDDK tag covalently attached to high-binding capacity magnetic agarose, we demonstrate effective purification of recombinant proteins expressed in bacteria, the Invitrogen™ ExpiCHO™ Expression System, and by a HeLa in vitro translation (cell-free) system. Biological activity was demonstrated for ExpiCHO-expressed DYKDDDDK–Tumor Necrosis Factor alpha (TNFα) which was used to successfully stimulate cells and activate Nuclear Factor kappa B (NFkB) p65. In addition, successful co-immunoprecipitation of DYKDDDDK-tagged BAD with 14-3-3 proteins was confirmed using an optimized sample preparation protocol in conjunction with the Thermo Scientific™ KingFisher™ Flex Magnetic Particle Processor and mass spectrometry. In summary, anti-DYKDDDDK magnetic agarose can be used to effectively isolate, purify, and enrich functional protein targets in a variety of proteomic applications.

Learning Objectives:

• Learn how to automate protein applications using cell-based and cell-free systems with magnetic beads