S代表样品制备（Sample Preparation）
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。
3. 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（Redondo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-PAGE
? Native PAGE or
? Dynamic Light Scattering
? IEF (Isoelectric Focusing) Gel
? Mass Spectroscopy
根据这些试验的结果，我们可以判断：
? 蛋白样品的纯度
? 蛋白样品的同次性
? 不同批次纯化出来的蛋白的性质差异
? 蛋白质的稳定性
5. 蛋白需要达到什么纯度？要拿去筛晶体，蛋白样品需要达到什么纯度？答案是越纯越好。但是这样一个答案不能让人满意。如何能更容易理解一些呢？一开始筛选时，根据考马斯亮蓝染色判断蛋白纯度应该达到90 到 95%。不过，考虑到总有进一步纯化的步骤，不可能达到‘绝对’的100%纯，所以没有必要追求‘绝对’纯，存在一点点的杂质蛋白是可以接受的。还要记住，结晶本身就是一个纯化过程。当筛选过程中没有晶体长出，甚至没有长的迹象，或者在优化条件时不能进一步提高晶体的质量了，这个时候就要考虑进一步提高蛋白浓度了。
6. 样品的保存。一般蛋白都可以在4°C 或 -70°C保存。与制备样品的人交流一下或者查阅文献中报道的类似蛋白的保存方法，选择一个合适的缓冲液体系和保存温度。在保存之前，应该测定蛋白的活性和稳定性，并隔一段时间取出一点蛋白来测定其活性是否改变及有没有降解发生。反复冻溶对蛋白是不利的，应尽量避免。样品应该分装成小份，每次取出当次试验需要的量，保证取出来的正好用完。有时候，一些人喜欢加入甘油（10 to 50% v/v）来保护蛋白不被冻伤。但是应该尽量避免加入甘油，因为甘油很难通过透析或者过滤来除干净，残存的甘油会给长晶体带来变数。甘油在不同条件下可能是沉淀剂，也可能是additive（促溶剂？）或冷冻保护剂。一般来讲，蛋白浓度越高越利于保存。但是，当蛋白浓度很高时，保存期间也容易产生沉淀。将保存的蛋白做好标记，是何批次纯化的，蛋白浓度多高，以及保存的时间等。将同一个批次的蛋白分装好保存到同一个大的离心管里，这样方便管理。
7. 蛋白样品操作中要注意的。Be nice to your protein.要记得蛋白可是微生物们的美餐，不要让它们偷吃了你的蛋白。平时，蛋白一定要在4° C或更低温度放置，暴露在室温的时间要尽可能地短。当溶化一个蛋白样品或将冷冻干燥的蛋白复溶的时候，不要摇晃或震荡，不要产生气泡，出现气泡往往标志着蛋白变性了。当确定了点晶体时操作间的温度，应预先将蛋白样品在这个温度放置一会儿。在蛋白结晶的领域里存在很多的不同的主张。比如，有人认为在点晶体之前应过滤蛋白除掉可能存在的细菌和蛋白聚集体及杂质等，而有人在认为不应该过滤或离心，那些并不明显的蛋白聚集体或杂质等可能是结晶所需的晶核。
8. 要不要加叠氮化钠？NaN3是一种有效抑制细菌污染的添加剂。常用的工作浓度为1 mM或0.02% 到 0.1% w/v.因为叠氮化钠有毒，所以操作时一定要小心。当决定添加叠氮化钠了，就需要注意以下几点：
? 它能杀菌，对人体也是有害的。
? 它是某些蛋白的抑制剂，可能影响试验。
? It can interfere with heavy atom derivatization.
? 一些叠氮化物是爆炸性的
? 有报道指出除掉叠氮化钠能促进晶体生长
可以替代叠氮化钠的试剂有麝香草酚（Thymol）和Thimerosal。还有其他的方法，那就是每一步操作时都保证仪器和器材（灭菌枪头等）都是无菌的，溶液都要过滤除菌，并在4°C或更低温度保存。所有这些细节都做好了，就可以避免使用化学添加剂来防菌了。
9. 试验记录要做好。纯化，保存，点晶体等每一个步骤都应该详细记录。筛晶体时各个条件也都要做好记录以备需要时查阅。这些需要记录的项目包括：
? 有关这个蛋白的信息
? 样品名称
? 样品的纯化批次，保存地点及时间，保存条件等
? 蛋白缓冲液的成分，添加剂浓度等
? 样品浓度
? 结晶试验的信息
? 方法
? 液滴大小及成分
? 池液的成分
? 温度
? 日期
? 实验者
乍看起来这有些过于拘泥细枝末节，但是要知道所记录下的这些信息都可能成为结晶筛选的一个变量。做详尽准确的试验记录是必要的。在记录结晶的情况时，一些描述性的和定量的记录都应该有。最后，还有保持操作间的整洁卫生，避免化学物质和细菌的污染。好的习惯让人受益。
10. 关于你的样品还应该掌握的信息：
? 是否有同源性很高的蛋白已经解出结构了？
? 样品中存在自由的半胱氨酸吗？
? 样品中有哪些添加剂？
? 样品上是否有糖基？
? 样品蛋白是否是磷酸化的？
? 样品N端是否甲基化？
? 样品在什么温度下稳定？
? 样品的活性和稳定性随温度如何改变？
? 样品的可溶性和稳定性随pH如何变化？
? 样品结合金属离子吗？
? 样品对蛋白酶敏感否？
? 我的样品属于酶，膜蛋白，抗体？
? 对这类的蛋白通常使用什么方法？
? 样品蛋白的来源？
? 蛋白样品到达我手上之前如何纯化和保存的？
? 样品的缓冲液体系？
? 是否同一批次纯化出来的？
? 我手上有多少蛋白？我还能获得多少？
? 蛋白纯度如何？
? 均一性同次性如何？
? 这个蛋白独特的性质是什么？
其他与蛋白质操作过程中的注意事项相关的日志：（Redondo推荐）
1. 如何避免蛋白发生聚集？
2. 使用Ni亲和层析柱纯化蛋白时需要注意的事项
3. 蛋白质操作过程中需要注意的问题
4. 关于蛋白稳定性需要考虑的几个因素
5. 蛋白表达与纯化总结PPT
6. 收藏：沉淀蛋白方法
7. 蛋白样品制备中需要注意的问题
附原文：
Preparing the Protein for Crystallization
Lyophilization
Avoid lyophilization. Even though there are many examples of proteins which crystallize after lyophilization (lysozyme, thaumatin, hemoglobin), lyophilization is to be avoided when possible. If the protein is lyophilized, it needs to be dialyzed before crystallization. Dialyze the protein against deionized water or a stabilization buffer before crystallization. Dialysis will remove non-volatile buffers and other chemicals which may have been present before lyophilization.
Ammonium Sulfate Precipitation
Avoid using ammonium sulfate precipitation as a final purification and/or concentration step. It is often very difficult to completely remove all the ammonium sulfate by a desalting column of dialysis. The remaining trace amounts of ammonium sulfate can interfere with crystallization screening results and create reproducibility problems. It is not uncommon for trace amounts of ammonium sulfate in the sample to cause precipitation or excessive nucleation in screen conditions containing polyethylene glycol and salt.
Batches
Avoid combining different purification batches for crystallization trials. Purification conditions and procedures are never identical so each batch should be screened separately.
Profile the Protein
Ideally, you will purify your own protein, but this is not always reality. So, it is always a good idea to characterize your protein before beginning crystallization experiments. Profiling your protein before crystallization can often provide valuable clues during screening and optimization of crystallization conditions. Assays to seriously consider:
? SDS-PAGE
? Native PAGE or
? Dynamic Light Scattering
? IEF (Isoelectric Focusing) Gel
? Mass Spectroscopy
The results of these assays can:
? Determine the purity of the sample
? Determine the homogeneity of the sample
? Identify batch-to-batch variations
? Identify stability problems with the sample
How Pure?
How pure should the protein sample be for crystallization trials? As pure as possible. That’s some answer, is it not? Integrating common sense into the question, we might arrive at the following answer. For initial screening, the sample should be at least 90 to 95% pure on a Coomassie stained SDS-PAGE. Finally, it does no harm to screen an “impure sample” as one can always perform further purification. Remember, crystallization used to be considered a very powerful purification tool (and still is!).
If the initial screen does not produce crystals, any promising results, or it becomes next to impossible to improve crystal quality during optimization, one should consider further purification of the sample.
Storing the Sample
Most proteins can be stored successfully at 4°C or -70°C.
Check with the person preparing the protein or compare your protein to a similar protein in the literature for best storage temperature.
Ideally, one should assay the activity and stability of the protein before storage and then later on at various points in time to determine the sample storage stability.
Repeated freezing and thawing of the sample should be avoided. Aliquot the sample into multiple small microcentrifuge tubes. Make the aliquots small enough so that the entire aliquot can be consumed in the experiment after thawing.
Sometimes people like to add glycerol (10 to 50% v/v) to help proteins better tolerate freezing. Avoid this if possible since it is often difficult to remove glycerol by dialysis or filtration. The presence of glycerol is a crystallization variable. Glycerol can behave as a precipitant, an additive, or cryoprotectant and therefore can influence the outcome of a crystallization experiment.
In general, it is better to store proteins more concentrated than diluted. When too dilute, adsorption of the protein onto the storage container can lead to significant losses. However, precipitation can sometimes be a problem when the protein is stored too concentrated.
Label samples clearly with the sample identification, batch identification, and date of storage. Small cryo labels can be very useful here. Color coding samples can be a nice organization tool. For the sake of easy organization and identification, it is sometimes more convenient to nest samples. For example, store batches of small microcentrifuge tubes in 10 ml or 50 ml centrifuge tubes and organize them by batch or sample.
Sample Handling
Be nice to your protein. Remember that proteins make an excellent food source for microbes. Protect your sample from microbes by storing the protein at less than 4° C and not leaving the sample for extended periods of time at room temperature.
When thawing a sample or mixing a lyophilized sample into solution do not shake or vortex the protein. Avoid foaming the sample. Foam can be a sign of denaturation.
Allow the sample to equilibrate to the temperature where the crystallization experiments will be set up and/or incubated before setting the experiment.
The field of crystal growth is full of opinions and controversy. There are several opinions on what should be done with the sample just prior to setting the crystallization experiment. Let’s have a look at those opinions.
Some like to filter the sample through a 0.2 micron (or smaller, but be sure to compare the MW of your sample to the pore size of your filter so as not to stick your sample on the filter) pore size filter into a sterile container. Filtration can remove microbial contamination (but not the proteases) as well as sample aggregation. Turbid sample solutions with lots of precipitate should be solubilized or centrifuged before filtration to avoid the ugly experience of sticking the sample to a filter membrane. Use filters with the smallest possible dead volume to minimize sample loss. Some of the centrifugal microfilration devices are certainly worth consideration. Before filtering the sample, wash or flush the filter with a small amount of the sample buffer / storage solution before filtering the sample. This will test the filter for compatibility with your sample buffer and remove any trace glycerol which can sometime be present from the manufacturer. If possible, test filter a small aliquot of the sample, and measure the activity / OD before filtering the entire sample. Do this to test the adherence of the sample to the filter media. Read and follow the instructions supplied before introducing the sample to the filter.
Some like to centrifuge the sample. Centrifugation removes large sample aggregates and amorphous debris. Post centrifugation views can provide a visual clue of aggregation/precipitate for seemingly clear solutions. Following centrifugation, use only the supernatant for crystallization trials.
Others prefer to avoid filtration or centrifugation before setting crystallization experiments. One view is that the presence of amorphous material or aggregates can enhance the changes for crystallization be acting as nucleants.
To Azide or Not
Sodium azide (NaN3) is an anti-microbial preservative that is sometimes used to protect samples and crystallization reagents from microbial contamination. Sodium azide is toxic and should be handled with care. Typical sodium azide concentrations are 1 mM or if you prefer % measurements, between 0.02% and 0.1% w/v.
If you choose to use sodium azide remember that:
? It is toxic to humans as well as microbes.
? It is an inhibitor for some proteins and may become an unintentional ligand for your sample.
? It can interfere with heavy atom derivatization.
? Some metal azides are explosive.
? There are reports where eliminating sodium azide from the experiment improved crystallization.
Alternatives to Sodium Azide Include Thymol and Thimerosal.
A final alternative to the use of antimicrobials is the use of proper sterile technique and materials. Sterile filter all samples and reagents into sterile containers. Store samples and reagents at 4°C or lower. Use sterile pipet tips. Keep your work area clean. Develop a sterile technique with your crystallization setups. With common sense, sterile reagents and sample, good technique, and sterile pipet tips, one can successfully avoid the use of chemical antimicrobials in the crystallization lab.
Record Keeping and Organization
It is prudent to write down and hold onto detailed notes concerning the purification, storage, and handling of the sample. It is obvious that one should also maintain records of crystallization trials which should include:
? Sample information
? Name of sample
? Sample identification (batch, storage location, storage temperature, etc)
? Sample buffer composition, additives, ligands, etc.
? Sample concentration
? Crystallization experiment information
? Method
? Drop size and composition
? Reagent composition
? Temperature
? Date
? Person performing experiment
Although it may seem trivial, a little AR, and excessive, it is reasonable to write down anything that could become a crystallization variable.
Maintaining accurate and complete records of experimental observations is obviously important and will be covered in more detail elsewhere. In general, try to use both descriptive and quantitative comments when developing crystallization records.
Maintain a clean and well organized workplace for your crystallization setups. Cleanliness will help to prevent chemical, microbial, and miscellaneous contamination. Organization will prevent errors and save time.
Questions to Ponder About the Sample
? Does a similar sample exist and has it been crystallized?
? Does the sample contain free cysteines?
? Does the sample contain additives such as sodium azide, ligands, inhibitors,
or substrates?
? Is the protein glycosylated?
? Is the protein phosphorylated?
? Is the protein N-terminal methylated?
? At what temperature is the protein stable?
? How does sample solubility and stability change with temperature?
? How does sample solubility and stability change with pH?
? Does the sample bind metals?
? Is the protein sensitive to proteolysis?
? What class of protein am I working with (antibody, virus, enzyme, membrane protein)?
? What have been the most successful approaches with my class of protein?
? What is the source of the sample?
? How was the sample purified and stored before it arrived into my hands?
? What is in the sample container besides the sample (buffer, additives, etc.)?
? Is the sample pooled purification aliquots or a single batch?
? How much sample do I have and how much more is available?
? How pure is the sample?
? How homogeneous is the sample?
? Does anyone possess any solubility information on this sample?
? What is unique about this protein?
? What is necessary chemically and physically to maintain a stable, active sample?
