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.
Catimel
B
B
The Ludwig Institute for Cancer Research, Melbourne Tumor Biology Branch, PO Box 2008, Royal Melbourne Hospital, Victoria 3050, Melbourne, Australia.
Rothacker
J
J
Catimel
J
J
Faux
M
M
Ross
J
J
Connolly
L
L
Clippingdale
A
A
Burgess
A W
AW
Nice
E
E
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
D015820:Cadherins; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D051176:beta Catenin; D012685:Sepharose; D005982:Glutathione Transferase