Catimel Bruno, Nice Edouard C, Kärrlander Maria, Ross Janine, Catimel Jenny, Burgess Antony W, Faux Maree
Ludwig Institute for Cancer Research, PO Royal Melbourne Hospital, Parkville, Victoria, Australia.
Biomed Chromatogr. 2006 Jun-Jul;20(6-7):569-75. doi: 10.1002/bmc.648.
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.
重组蛋白通常与亲和标签融合表达以利于纯化，常被用作多克隆抗体制备的免疫原。对抗体产物进行仔细的免疫纯化往往是获得针对目标蛋白结构域的高特异性多克隆抗体的关键。本研究描述了一种针对腺瘤性息肉病大肠杆菌（APC）肿瘤抑制因子的抗体的纯化及特性鉴定。我们结合使用亲和色谱法和生物传感器分析来优化和监测抗体纯化过程。然后通过免疫沉淀、蛋白质组学分析和免疫荧光染色对该抗体进行特性鉴定，结果表明它是研究APC生物学的一种有价值的试剂。利用该抗体，我们通过串联质谱（MS/MS）成功地从转染细胞系中分离并鉴定出了APC。在APC的丝氨酸1436位点鉴定出了一个新的磷酸化位点。涉及多个免疫亲和步骤并结合表面等离子体共振（SPR）、免疫沉淀蛋白质组学和免疫荧光分析的类似策略通常适用于其他多克隆抗体的纯化及特性鉴定。
