Pietrocola Giampiero, Rindi Simonetta, Nobile Giulia, Speziale Pietro
Unit of Biochemistry, Department of Molecular Medicine, University of Pavia, Pavia, Italy.
Methods Mol Biol. 2017;1627:309-324. doi: 10.1007/978-1-4939-7113-8_20.
A method is described for the purification of plasma fibronectins based on a combination of gelatin- and arginine-Sepharose chromatography steps. Cellular fibronectin can be purified from an osteosarcoma fibroblast cell line by affinity chromatography using a monoclonal antibody anti-fibronectin as ligand. Furthermore, we also provide a protocol for the purification of fibronectin domains obtained by fractionation of thermolysin-digested plasma fibronectin on ion-exchange/gel filtration chromatography columns. Assessment of the fibronectin purity is performed by SDS-PAGE, while the ligand binding activities of specific fibronectin domains are determined by ELISA.
描述了一种基于明胶和精氨酸-琼脂糖层析步骤相结合的血浆纤连蛋白纯化方法。细胞纤连蛋白可通过使用抗纤连蛋白单克隆抗体作为配体的亲和层析从骨肉瘤成纤维细胞系中纯化得到。此外，我们还提供了一种通过在离子交换/凝胶过滤层析柱上对嗜热菌蛋白酶消化的血浆纤连蛋白进行分级分离来纯化纤连蛋白结构域的方案。通过SDS-PAGE评估纤连蛋白的纯度，而通过ELISA测定特定纤连蛋白结构域的配体结合活性。
