Homandberg G A, Evans D B, Kramer J, Erickson J W
J Chromatogr. 1985 Jun 26;327:343-9. doi: 10.1016/s0021-9673(01)81663-0.
Fibronectin is a large, adhesive glycoprotein which self-associates on many cell surfaces. We have begun to study this reaction by determining the domains of fibronectin which interact with each other. To avoid possible solid-phase artifacts of affinity chromatography, we have devised a solution-phase assay in which the smallest fibronectin fragment is labeled with fluorescamine, mixed with unlabeled fibronectin, and complexation is observed by the appearance of a new higher-molecular-weight peak on gel high-performance liquid chromatography columns. The assay allowed use of excess unlabeled reactant, high-sensitivity, low background without removal of reagent, and fast analysis. Our results show that the amino- and carboxyl-terminal fibronectin fragments bind the native molecule in solution.
纤连蛋白是一种大型的黏附糖蛋白，它在许多细胞表面进行自我缔合。我们已开始通过确定纤连蛋白中相互作用的结构域来研究这一反应。为避免亲和色谱可能出现的固相亲和假象，我们设计了一种溶液相分析方法，其中最小的纤连蛋白片段用荧光胺标记，与未标记的纤连蛋白混合，通过凝胶高效液相色谱柱上出现新的更高分子量峰来观察络合情况。该分析方法允许使用过量的未标记反应物，具有高灵敏度、低背景且无需去除试剂，分析速度快。我们的结果表明，纤连蛋白的氨基末端和羧基末端片段在溶液中与天然分子结合。
