Biosensor-Based Micro-Affinity Purification for the Proteomic Analysis of Protein Complexes
| Autor: | Anthony W Burgess, A Clippingdale, Jenny Catimel, Julie Rothacker, Bruno Catimel, Lisa M. Connolly, Janine Ross, Maree C. Faux, Edouard C. Nice |
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| Rok vydání: | 2005 |
| Předmět: |
Proteomics
Time Factors Blotting Western Biosensing Techniques Biology Biochemistry Mass Spectrometry law.invention Sepharose Affinity chromatography law Cell Line Tumor Humans Phosphorylation Polyacrylamide gel electrophoresis beta Catenin Glutathione Transferase Chromatography Proteins General Chemistry Cadherins Chromatography Ion Exchange Ligand (biochemistry) Recombinant Proteins Protein Structure Tertiary Recombinant DNA Electrophoresis Polyacrylamide Gel Peptides Biosensor Chromatography Liquid Protein Binding Binding domain |
| Zdroj: | Journal of Proteome Research. 4:1646-1656 |
| ISSN: | 1535-3907 1535-3893 |
| Popis: | 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−β-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. Keywords: biosensor micro-affinity purification • proteomics • APC • E-cadherin • β-catenin |
| Databáze: | OpenAIRE |
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