Catimel B, Rothacker J, Catimel J, Faux M, Ross J, Connolly L, Clippingdale A, Burgess A W, Nice E
The Ludwig Institute for Cancer Research, Melbourne Tumor Biology Branch, PO Box 2008, Royal Melbourne Hospital, Victoria 3050, Melbourne, Australia.
J Proteome Res. 2005 Sep-Oct;4(5):1646-56. doi: 10.1021/pr050132x.
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.
已开发出一种基于生物传感器的微亲和纯化方法，用于回收蛋白质结合伴侣及其复合物，以便进行下游蛋白质组学分析。该方法使用配备原型表面处理单元（SPU）的BIAcore 3000，将重组E-钙黏蛋白的GST-细胞内结构域或重组腺瘤性息肉病大肠杆菌（APC）的GST-β-连环蛋白结合结构域固定在SPU上，并用于从经色谱富集的SW480结肠癌细胞裂解物中亲和纯化结合伴侣。以固定有GST的表面作为对照。从SPU回收的样品进行SDS-PAGE和灵敏考马斯亮蓝染色，然后进行自动胶内消化和LC-MS/MS。将使用SPU获得的结果与使用琼脂糖珠进行的类似实验结果进行比较。
