5:30 – 8:30 pm
(SC10) AFFINITY TAGS FOR PROTEIN PURIFICATION
Dinner, Presentations & Interactive Discussion
- Overview of Affinity Purification
- Types of Affinity Tags
- Comparison of Affinity Tags
- Tag Cleavage/Self-Cleaving Tags
- Alternative Tagging Strategies
- Streamlining Protein Recovery
- Emerging Technologies
5:30 Chairperson’s Remarks
John Proctor, Ph.D., Product Manager, Biosensors and Assay Kits, FortéBio, Inc.
5:40 Choosing the Right Tag: For You and Your Protein
 William Gillette, Ph.D., Senior Scientist, Protein Expression Lab, SAIC-NCI Frederick
Affinity tags offer a wide array of features and drawbacks for both protein expression and purification. A discussion of the pros and cons of these tags will be followed by examples of how their use simplifies (or complicates) protein expression and purification in a Core Service lab.
6:45 Q&A -- Discussions
7:00 Dinner Break
7:30 Using Tandem Affinity Purification-Mass Spectrometry for Analyzing Intracellular Signaling Complexes
 Alexey Veraksa, Ph.D., Assistant Professor, Biology Department, University of Massachusetts, Boston
In this talk, I will use a case study of mapping the protein complexes in the Notch pathway to share our experiences with tandem affinity purification (TAP)-mass spectrometry approaches. The Notch signaling pathway is a conserved mechanism of intercellular communication that is used in all metazoans to establish molecular differences between adjacent cells. We have used the original TAP tag, as well as more recently developed GS-TAP tag, to analyze protein interactions in the Notch pathway. We have validated some of the novel interactions using functional assays in cultured cells and in vivo. I will discuss the advantages and pitfalls associated with the use of the TAP tags, and will share our new data on developing even more efficient ways to isolate and analyze protein complexes.
8:30 Close of Affinity Tags for Protein Purification Short Course
Speaker Bio Sketches
Dr. William Gillette
The Protein Expression Laboratory provides protein expression/purification core service to the laboratories of the NCI, NIH and USAMRIID. Dr. Gillette is currently focusing on micro-scale purification and supporting analysis techniques to evaluate protein expression constructs and the success of the micro-scale chromatography experiments. Dr. Gillette earned his Doctorate in Microbiology from North Carolina State University, Raleigh, North Carolina.
Dr. Alexey Veraksa
My laboratory studies signaling networks in Drosophila development, with a focus on dynamic protein-protein interactions that control developmental signaling pathways. As a postdoctoral fellow at the MGH Cancer Center, Harvard Medical School, I applied new protein purification methods in Drosophila and used them in combination with mass spectrometry to analyze protein complexes in the Notch interaction network. This study identified the Drosophila β-arrestin Kurtz (Krz) as a Notch signaling modulator that regulates the turnover of the Notch receptor. After becoming an independent researcher at UMass Boston, I continue to investigate the diverse functions of β-arrestin Krz in Drosophila development. We have recently found that Krz inhibits MAPK signaling at multiple developmental stages by a previously unknown mechanism. Part of our research efforts is devoted to developing new protein purification strategies that can be used for isolating and characterizing protein complexes involved in signal transduction. I received my Doctorate in Developmental Biology from the University of California, San Diego in La Jolla, California.
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