本研究以金奈米粒子做為固態載體，在金奈米上進行表面修飾，製備成金奈米生物探針，並且將此探針應用在免疫沉澱(Immunoprecipitation, IP)實驗上，對乳癌細胞MCF-7細胞萃取液中的雌激素受體阿爾法蛋白質(estrogen receptor alpha, ERα)進行免疫沉澱。將蛋白質G修飾之金奈米粒子與抗體Fc端形成鍵結，形成具有方向性的雌激素受體阿爾法抗體(anti-estrogen receptor alpha; anti-ERα )探針。為了進一步降低金奈米探針的非專一吸附，我們以相同方法合成修飾IgG抗體金奈米探針，作為預清理的探針，將細胞中會造成非專一性吸附的蛋白質去除，較乾淨的上清液再和修飾雌激素受體抗體金奈米探針進行免疫沉澱。結合預清理的步驟和抗體金奈米探針免疫沉澱，從MCF-7細胞萃取液中進行免疫沉澱，由一盤15公分細胞培養皿所得到的雌激素受體阿爾法的回收率約為81%，絕對量約為10 pmol。與市售的用於免疫沉澱的固態載體膠體小珠相比之下，抗體金奈米探針具有較高的雌激素受體阿爾法回收率且較低對serum albumin、HSP、ribosomal proteins等高存在量之非專一性蛋白質吸附。直接經由質譜分析，不用再經過SDS-PAGE分離，即可鑑定到目標蛋白質ERα。以膠內消化的方式再配合多種酵素組合，鑑定到的雌激素受體阿爾法序列涵蓋率為71%，且不需要經過額外的親和層析純化方法，也可觀察到雌激素受體阿爾法上某些具有轉譯後修飾的勝肽序列。

In this study, we fabricated immunoprobes using gold nanoparticles (AuNPs) as the solid support and applied the probes for immunoprecipitation against estrogen receptor alpha (ERα) in MCF-7 breast cancer cell lysates. We used protein G modified AuNPs to bind with the Fc domain of anti-ERα for ERα purification. In order to reduce the non-specific binding, we used the same method to fabricate AuNP-based anti-IgG probes for sample pre-cleaning prior to IP experiments. Combining the pre-cleaning step using AuNP-IgG probe and IP experiments using AuNP-anti- ERα probe, we determined the absolute amount of ERα from a 15-cm dish of MCF-7 cells was around 10 pmol and the IP recovery was around 81%. Compared to the commercial gel beads used in IP experiment, the AuNPs-based probes have higher recovery rate but much less non-specific binding towards many high abundant proteins such as serum albumin, HSP, and ribosomal proteins. Based on MS analysis of the pulled down proteins, the targeting protein ERα could be identified without separation by SDS-PAGE. By coupling SDS-PAGE separation and multiple enzyme digestion, a sequence coverage of 71% ER could be achieved and some phosphorylation and oxidations sites of ERα could be positively identified without additional affinity purifications.

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