Olman M A, Williams W F, Strickland J H, Hagood J S, Simmons W L, Rivera K E
Department of Medicine in the Division of Pulmonary and Critical Care Medicine, University of Alabama, Birmingham, Alabama, 35294, USA.
Protein Expr Purif. 1998 Oct;14(1):71-8. doi: 10.1006/prep.1998.0911.
We and others have recently shown that specific fragments of cross-linked fibrin affect cell behavior. In order to develop a facile method for the preparative scale purification of fibrin fragment D dimer, a simple gradient generating system for conventional chromatography was developed and validated, and methods of fibrin fragment D dimer purification were compared. The experimentally measured salt concentration/time relationship fell directly on the model-predicted line. Model-predicted changes in the reservoir volume and/or salt concentration in the limit buffer affected both the initial slope and the shape of the concentration/time relationship. This gradient generation method was used to separate the D domains of fibrin(ogen) from the amino terminal region E domain using anion-exchange chromatography. While the predicted salt gradient was achieved, a salt-dependent separation was found to be less optimal than that of a pH-dependent separation, as validated by Coomassie-stained SDS-PAGE and by immunoblotting. In conclusion, a facile, user-friendly, computer-based method to predict and generate salt gradients was written and validated by direct experimentation. While fibrinogen fragment purification was acceptable using this system, both separation and yields of fibrinogen and fibrin fragments were superior using a pH-based separation technique.
我们和其他研究人员最近表明，交联纤维蛋白的特定片段会影响细胞行为。为了开发一种用于制备规模纯化纤维蛋白片段D二聚体的简便方法，我们开发并验证了一种用于常规色谱的简单梯度生成系统，并比较了纤维蛋白片段D二聚体的纯化方法。实验测量的盐浓度/时间关系直接落在模型预测线上。模型预测的极限缓冲液中储液器体积和/或盐浓度的变化影响了浓度/时间关系的初始斜率和形状。这种梯度生成方法用于通过阴离子交换色谱从氨基末端区域E结构域中分离纤维蛋白（原）的D结构域。虽然实现了预测的盐梯度，但通过考马斯亮蓝染色的SDS-PAGE和免疫印迹验证，发现盐依赖性分离不如pH依赖性分离理想。总之，我们编写了一种简便、用户友好、基于计算机的预测和生成盐梯度的方法，并通过直接实验进行了验证。虽然使用该系统纯化纤维蛋白原片段是可以接受的，但使用基于pH的分离技术时，纤维蛋白原和纤维蛋白片段的分离和产量都更高。
