小中大4. Bulk or Batch Procedures for Purification
These procedures are almost always utilized early in the purification as they are usually most effective in removing non protein material and are amenable to the relatively large volume and amounts of material that exist in earlier stages of the preparation. A great deal of effort went into designing these steps in the early days of protein chemistry, and much frustration, time, and money can often be avoided by including some of these ‘‘old fashioned’’ procedures.
大量或批量蛋白纯化的步骤
纯化早期,常用这些手段来有效去除非蛋白类物质。在制备初期,大体积样品,高非蛋白物质含量用这些手段常常很实用。在蛋白质化学的早期阶段,前人做了大量的工作来完善这些批量纯化手段,那些费力、费时间和费钱的纯化过程如果包括这些“老式”的纯化手段常常可以得到改善。
Section V details many of these procedures. Gentle procedures including ‘‘salting out’’ or phase partition with organic polymers are most common. More drastic methods such as heat, extremes of pH, or phase partition with organic solvents might be particularly effective with stable proteins, though subtle forms of damage may be difficult to foresee or to detect. In addition, one might consider the use of high-capacity ion-exchange resins either added as a slurry to crude material or used in columns for batch elution. The time expended in developing and optimizing these early steps is always worthwhile—even removal of half of the contaminating protein may dictate the feasibility of a subsequent step from both cost and technical considerations.
第五节详述了这些“老式”的方法。温和的方法常用的有用有机高分子聚合物的“盐出”或相分配。更为剧烈的方法,例如加热、极端pH,或用有机溶剂的相分配,对某些稳定性的蛋白可能特别有效,尽管细微的损害难以预测或检测到。另外,可以考虑应用高容量的离子交换介质,可以直接作为胶浆加入到粗原料中,或者装柱做批次纯化洗脱。花时间研究优化这些早期步骤往往很有回报,即使只去除了一半的杂质蛋白,对后续步骤的成本和技术考虑都是很有益。
对头。对纯化来说,技术新,技术高并不代表是实用的好技术。这一点往往被我们忽视了。做蛋白质纯化,现在的试验室动则HPLC,制备电泳,密度离心,SOURCE,RESOURCE,用高分辨率的仪器,用高分辨率的纯化介质等等。不选对的,只选贵的。一个纯化工艺中如果有着很普通的胶,很简单的加热、沉淀等手段好像就不能显示纯化水平了,文章就低档次了,唉。
如果能根据目标蛋白的性质,选取用到有机溶剂萃取、沉淀(pH、硫酸铵、PEG等)、热处理等简单的方法对纯化大大有益。因为这些都是能够很方便放大的工艺方法。