Kim Sura, Ku Jayoung, Park Jaemin, Kharbash Raisa, Li Sheng
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST).
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST); KI for Health Science and Technology (KIHST), Korea Advanced Institute of Science and Technology (KAIST).
J Vis Exp. 2018 Nov 19(141). doi: 10.3791/58843.
We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltriethoxysilane (APTES) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTES. The deposition of APTES and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.
我们展示了一种制备用于抗体固定及后续免疫沉淀（IP）应用的聚（五氟苯基丙烯酸酯）（聚（PFPA））接枝二氧化硅微珠的简单方法。聚（PFPA）接枝表面通过一个简单的两步过程制备。第一步，将3-氨丙基三乙氧基硅烷（APTES）作为连接分子沉积到二氧化硅表面。第二步，通过可逆加成-断裂链转移（RAFT）聚合合成的聚（PFPA）均聚物，通过聚合物上的五氟苯基（PFP）单元与APTES上的胺基之间的交换反应接枝到连接分子上。通过X射线光电子能谱（XPS）确认了APTES和聚（PFPA）在二氧化硅颗粒上的沉积，并通过动态光散射（DLS）测量的粒径变化进行监测。为了提高微珠的表面亲水性，还进行了用胺官能化聚乙二醇（氨基-PEG）对聚（PFPA）的部分取代。然后将PEG取代的聚（PFPA）接枝二氧化硅微珠固定抗体用于IP应用。为了进行演示，使用了一种针对蛋白激酶RNA激活（PKR）的抗体，并通过蛋白质印迹法测定IP效率。分析结果表明，固定抗体的微珠确实可用于富集PKR，而非特异性蛋白质相互作用最小。
