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Biosensor-based micro-affinity purification for the proteomic analysis of protein complexes.

B Catimel·J Rothacker·J Catimel·M Faux·J Ross·L Connolly·A Clippingdale·A W Burgess·E Nice
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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.

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Biosensing TechniquesBlotting, WesternCadherinsCell Line, TumorChromatographyChromatography, Ion ExchangeChromatography, LiquidElectrophoresis, Polyacrylamide GelGlutathione TransferaseHumansMass SpectrometryPeptidesPhosphorylationProtein BindingProtein Structure, TertiaryProteinsProteomicsRecombinant ProteinsSepharoseTime Factorsbeta CateninJournal ArticleResearch Support, Non-U.S. Gov't

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B Catimel, J Rothacker, J Catimel, M Faux, J Ross, L Connolly, A Clippingdale, A W Burgess, E Nice. Biosensor-based micro-affinity purification for the proteomic analysis of protein complexes.. https://doi.org/10.1021/pr050132x

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