蛋白结晶的样品准备（Sample preparation for protein crystallization）.docVIP

蛋白结晶的样品准备（Sample preparation for protein crystallization） 的代表样品制备（样品制备） 1。避免冷冻干燥。尽管有一些蛋白是经冷冻干燥处理后长出了晶体，但是大多数情况下并非如此。所以要尽可能避免冷冻干燥。如果蛋白已经这样了，准备点晶体之前，要用水或者缓冲液透析，除掉非挥发性的试剂和其他化学物质。 2。避免硫酸铵沉淀。避免硫酸铵沉淀法作为纯化的最后一步或使用硫酸铵沉淀法来浓缩蛋白。因为硫酸铵很难除干净，对后续的结晶过程造成干扰。残存的微量的硫酸铵也会干扰晶体筛选的条件，造成结果的不可重复性。微量的硫酸铵也容易造成样品沉淀，以及在PEG和盐类物质存在时会出现过量的晶核。 三.蛋白批次。在同一次条件筛选时，要避免样品不是同一批次纯化出来的。每一次纯化的条件和步骤都是不同的［就像天下没有两片相同的树叶（雷东多添加）］，所以筛选的时候应该使用同一批次纯化出来的蛋白。 4。定性蛋白质。在拿去点晶体之前，应该定性判断你纯化出来的蛋白是不是你的目的蛋白。根据确定了的蛋白质性质，可以给筛选和优化提供参考。常用的蛋白质定性试验包括： ·SDS ·本地页或 动态光散射 ·IEF（等电聚焦）凝胶 ·质谱 Judging from the results of these tests, we can judge: Protein sample purity - protein sample homogeneity Differences in the properties of proteins purified in different batches Protein stability What purity does 5. protein need to achieve? What is the purity of the protein sample to be screened? The answer is, the purer, the better. But such an answer is not satisfactory. How can it be easier to understand? First screening, according to the Coomassie blue staining to determine the purity of protein should reach 90 to 95%. However, taking into account the total purification step, it is impossible to achieve absolute 100% pure, so there is no need to pursue absolute pure, there is a little impurity protein is acceptable. Bear in mind that crystallization itself is a purification process. When the screening process is not crystal out, and even without long signs, or in the optimization conditions can not further improve the quality of the crystal, this time we should consider further improve the protein concentration. 6. preservation of samples. General proteins can be stored at 4 degrees C or -70 degrees C. Communicate with the sample maker or consult the preservation methods of similar proteins reported in the literature, and select a suitable buffer system and storage temperature. Before preservation, the activity and stability of the protein should be measured, and a bit of protein will be removed for a period of time to determine whether the activi