Purification and characterization of a high specificity polyclonal antibody to the adenomatous polyposis coli tumour suppressor protein.
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.
Catimel
Bruno
B
Ludwig Institute for Cancer Research, PO Royal Melbourne Hospital, Parkville, Victoria, Australia.
Nice
Edouard C
EC
Kärrlander
Maria
M
Ross
Janine
J
Catimel
Jenny
J
Burgess
Antony W
AW
Faux
Maree
M
D025601:Adenomatous Polyposis Coli Protein / Q000276:immunology*; D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002460:Cell Line; D002846:Chromatography, Affinity; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D040901:Proteomics; D020349:Surface Plasmon Resonance
D025601:Adenomatous Polyposis Coli Protein; D000906:Antibodies