Biosensor-based micro-affinity purification for the proteomic analysis of protein complexes.
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.
Mesh Terms:
Biosensing Techniques, Blotting, Western, Cadherins, Cell Line, Tumor, Chromatography, Chromatography, Ion Exchange, Chromatography, Liquid, Electrophoresis, Polyacrylamide Gel, Glutathione Transferase, Humans, Mass Spectrometry, Peptides, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Proteins, Proteomics, Recombinant Proteins, Sepharose, Time Factors, beta Catenin
Biosensing Techniques, Blotting, Western, Cadherins, Cell Line, Tumor, Chromatography, Chromatography, Ion Exchange, Chromatography, Liquid, Electrophoresis, Polyacrylamide Gel, Glutathione Transferase, Humans, Mass Spectrometry, Peptides, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Proteins, Proteomics, Recombinant Proteins, Sepharose, Time Factors, beta Catenin
J. Proteome Res.
Date: Oct. 11, 2005
PubMed ID: 16212417
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