title: Biosensor-based micro-affinity purification for the proteomic analysis of protein complexes.
abstract: A biosensor-based micro-affinity purification method to recover protein binding partners and their complexes for down stream proteomics analysis has been developed using the BIAcore 3000 fitted with a prototype Surface Prep Unit (SPU). The recombinant GST-intracellular domain of E-cadherin or the recombinant GST-beta-catenin binding domain of Adenomatous Polyposis Coli (APC) were immobilized onto the SPU and used to affinity purify binding partners from chromatographically enriched SW480 colon cancer cell lysates. A GST- immobilized surface was used as a control. Samples recovered from the SPU were subjected to SDS-PAGE with sensitive Coomassie staining followed by automated in-gel digestion and LC-MS/MS. The results obtained using the SPU were compared with similar experiments performed using Sepharose beads.
authors.affiliation: The Ludwig Institute for Cancer Research, Melbourne Tumor Biology Branch, PO Box 2008, Royal Melbourne Hospital, Victoria 3050, Melbourne, Australia.
authors.forename: B
authors.initials: B
authors.lastname: Catimel
authors.forename: J
authors.initials: J
authors.lastname: Rothacker
authors.forename: J
authors.initials: J
authors.lastname: Catimel
authors.forename: M
authors.initials: M
authors.lastname: Faux
authors.forename: J
authors.initials: J
authors.lastname: Ross
authors.forename: L
authors.initials: L
authors.lastname: Connolly
authors.forename: A
authors.initials: A
authors.lastname: Clippingdale
authors.forename: A W
authors.initials: AW
authors.lastname: Burgess
authors.forename: E
authors.initials: E
authors.lastname: Nice
mesh_terms: D015374:Biosensing Techniques; D015153:Blotting, Western; D015820:Cadherins / Q000737:chemistry; D045744:Cell Line, Tumor; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry; D012685:Sepharose / Q000494:pharmacology; D013997:Time Factors; D051176:beta Catenin / Q000737:chemistry
chemical_list: D015820:Cadherins; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D051176:beta Catenin; D012685:Sepharose; D005982:Glutathione Transferase