Tandem Affinity Purification Tag.ppt.pptVIP

Background Information Rationale for Using TAP/MS Method The Tandem Affinity Purification Tag TAP Purification Strategy TAP Purification Strategy TAP Purification Strategy TAP Purification Strategy … in detail The Experiment The Experiment …cont’d Some Statistics A Higher-Order Map Linked Complexes Results of the Experiment Some Faults of the TAP Method Conclusions Great Summer Reading * Functional organization of the yeast proteome by systematic analysis of protein complexes Anne-Claude Gavin et al. Anne-Claude Gavin, Markus Bosche, Roland Krause, Paola Grandi, Martina Marzioch, Andreas Bauer, Jorg Schultz, Jens M. Rick, Anne-Marie Michon, Cristina-Maria Cruciat, Marita Remor, Christian Hofert, Malgorzata Schelder, Miro Brajenovic, Heins Ruffner, Alejandro Merino, Karin Klein, Manuela Hudak, David Dickson, Tatjana Rudi, Volker Gnau, Angela Bauch, Sonja Bastuck, Bettina Huhse, Christina Leutwein, Marie-Anne Heurtier, Richard R. Copley, Angela Edelmann, Erich Querfurth, Vladimir Rybin, Gerard Drewes, Manfred Raida, Tewis Bouwmesster, Peer Bork, Bertrand Seraphin, Bernhard Kuster, Gitte Neubauer, and Giulio Superti-Furga (Nature 415. 2002) Proteins rarely act alone Comprehensive protein interaction studies thus far: two-hybrid systems (ex vivo) protein chips (in vitro) GST pull-downs (in vivo) 3. Authors used tandem-affinity purification (TAP) and mass spectrometry Fast purification with high yield In vivo Over-expression of proteins not required Prior knowledge of protein complex not required Purified complex can be used in a variety of studies target protein target protein PCR of the TAP cassette Transformation of yeast cells (homologous recombination) Selection of positive clones Large-scale cultivation Cell lysis Tandem affinity purification One-dimensional SDSMALDI-TOF protein identification Bioinformatic data interpretation Processed 1,739 genes (1,143 human orthologues) Purified