Purification and characterization of a high specificity polyclonal antibody to the adenomatous polyposis coli tumour suppressor protein

Abstract

Recombinant proteins, commonly expressed in fusion with an affinity tag to facilitate purification, are often used as immunogens for polyclonal antibody production. Careful immunopurification of the antibody product is often the key to obtaining a high-specificity polyclonal antibody against the protein domain of interest. This study describes the purification and characterization of such an antibody directed against the adenomatous polyposis coli (APC) tumour suppressor. We used a combination of affinity chromatography and biosensor analysis to optimize and monitor antibody purification. This antibody was then characterized by immunoprecipitation, proteomic analyses and immunofluorescence staining and shown to be a valuable reagent for the study of APC biology. Using this antibody we successfully isolated and identified APC, using MS/MS, from transfected cell lines. A novel phosphorylation site on APC was identified at ser 1436. Similar strategies involving multiple immuno-affinity steps coupled with surface plasmon resonance (SPR), immunoprecipitation proteomic and immunofluorescence analyses should be generally applicable for the purification and characterization of other polyclonal antibodies.

Recruitment of adenomatous polyposis coli and β-catenin to axin-puncta

“…The following antibodies were used: anti-APC (APC2, produced in our laboratory (Catimel et al, 2006)), anti-APC (H290; Santa Cruz, Santa Cruz, CA, USA), anti-CK1a (Santa Cruz), anti-axin-1 (Zymed, San Francisco, CA, USA), anti-phospho-b-catenin (Cell Signaling, Danvers, MA, USA, 9561, pS33, pS37, pT41 and 9565, pT41, pS45), anti-b-catenin (19920/610153; BD Transduction Laboratories, San Jose, CA, USA), anti-GSK3b (G22320; BD Transduction Laboratories), anti-actin (clone AC-40; Sigma, St Louis, MS, USA), anti-btubulin (clone 2.1; Sigma) and anti-HA (262K#2362; Cell Signaling). To generate C-terminally tagged APC-GFP, enhanced GFP was subcloned as a BamH1/Not1 fragment into pEF-mycAPC (Faux et al, 2004) after removing the STOP.…”

Supplementary Methods and Materials from PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

“…Immunopurified polyclonal anti-Phlda1 antibody was generated for use on mouse tissue (1). Briefly, antibody was purified from rabbit serum immunized with C-terminal Phlda1 peptide (LHSQPQGHRLLRSC) (Auspep) conjugated to maleimide-activated keyhole limpet hemocyanin (Pierce).…”