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Immunopurification of Adenomatous Polyposis Coli (APC) Proteins
source: BMC research notes
year: 2013
authors: Kerryn L. Elliott, Bruno Catimel, Nicole L. Church, Janine L. Coates, Meredith J. Layton, Maree C. Faux, Antony W. Burgess
summary/abstract:BACKGROUND : The adenomatous polyposis coli (APC) tumour suppressor gene encodes a 2843 residue (310 kDa) protein. APC is a multifunctional protein involved in the regulation of β-catenin/Wnt signalling, cytoskeletal dynamics and cell adhesion. APC mutations occur in most colorectal cancers and typically result in truncation of the C-terminal half of the protein.
RESULTS : In order to investigate the biophysical properties of APC, we have generated a set of monoclonal antibodies which enable purification of recombinant forms of APC. Here we describe the characterisation of these anti-APC monoclonal antibodies (APC-NT) that specifically recognise endogenous APC both in solution and in fixed cells. Full-length APC(1-2843) and cancer-associated, truncated APC proteins, APC(1-1638) and APC(1-1311) were produced in Sf9 insect cells.
CONCLUSIONS : Recombinant APC proteins were purified using a two-step affinity approach using our APC-NT antibodies. The purification of APC proteins provides the basis for detailed structure/function analyses of full-length, cancer-truncated and endogenous forms of the protein.
organization: Ludwig Institute for Cancer ResearchDOI: 10.1186/1756-0500-6-429
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