蛋白质与蛋白组学

3. Amount of target protein or total activity (mg or units).
If there is a suitable enzyme assay for the target protein, it should be carried out on material from each major step. If the protein is not an enzyme or there is no quantitative assay, and if the protein is visible on a Coomassie blue stained SDS–PAGE, then often the stained gel is scanned and the amount of protein in the target protein band is determined. In other words, purity is determined and multiplied by the total protein to give an estimate of the total target protein.

目标蛋白总量或总活性（mg或单位）
如果目标蛋白有适合的酶分析方法，对每一个主要步骤都应该计算出量是多少。如果目标蛋白不是酶，或者没有适合的定量分析方法，若目标蛋白在考马斯亮蓝染色的SDS-PAGE电泳胶上可见，染色胶经常要做扫描分析，从而得到目标蛋白条带的蛋白量。换而言之，蛋白纯度由估计的总目标蛋白量与总蛋白量的比值来确定。

一般来说，用活性定量方法比较好。电泳扫描方便，但人为因素太大，可信度不高。所以，做蛋白纯化，对外可以有一个电泳扫描纯度图，让别人看看罢了，但自己一定要知道，这个纯度不可信。

4. Specific activity (units/mg).
If enzyme activity assays are possible, then the total activity (units) is divided by the total protein (mg) to give specific activity as units/mg.

比活性（U/mg）
如果可以测定酶活性，那么用总活性除以总蛋白量就可以得到比活性units/mg。

比活性是一个可以表征目标蛋白纯度的值，有时候比纯度更能说明纯化过程的优劣。它不仅说明了纯度如何，而且表现了纯化过程中目标蛋白活性的保持状况。

5. Overall yield (%).
The yield at a step in the procedure is the amount of target (either total target protein or total activity) at that step divided by the amount of target in the first step (defined as 100%).

总收率（%）
纯化过程每一步的收率是用此步所得到的目标蛋白量（总目标蛋白或总活性），除以前一步的目标蛋白量（定义为100%）而得到。

这个没什么好说的，要注意的就是活性收率可能存在大于100%的情况。这种情况时有发生。有时候是测定误差造成，但有时候不是测定误差的原因，而是在纯化过程中去除了某些可能影响活性的杂质而导致总活性增加，属于正常情况。

6. Purity of target protein (%).
Purity is often determined by scanning a stained SDS–PAGE and measuring the amount of the stain associated with the target band as a fraction of the stain associated with all the bands on the gel. If one has a reliable assay, then if the final material is pure, its specific activity can be used to define purity. For example, if an earlier step has a specific activity 10% of the final pure material, then the purity at that step would be 10%.

目标蛋白纯度（%）
纯度经常用SDS-PAGE电泳胶扫描来得到。由目标蛋白染色条带的扫描值比上胶上全部染色条带的扫描值得出。如果有可以信赖的分析方法，而最终的纯化产物是纯的，比活性可以用来定义纯度。例如，如果某一步的比活性为最终产物活性的10%，那么此步物料的纯度可以认为是10%。

呵呵，凝胶扫描前面说了有误差，受人为因素影响大。用活性来定义纯度也同样有着一些局限性。例如前一段所说的活性增加的情况，例如活性减少的情况等等。用活性来定义纯度要求，一是活性检测方法可信度高，系统方法误差小，二是体系中纯目标蛋白的比活性维持不变。实际上这两点都是很难达到的。
绕啊绕，又回到了原处。那就是无论活性还是纯度都是一个相对准确的定量值，受人为影响大啊。所以，做蛋白纯化还是要看最终的用途是什么。

7. Relative or fold purification.
This is not essential since it can be calculated from the other values above, but it is often useful. This is merely setting the initial purity at a value of one and then giving the purity at each step relative to that of the first step. For example, in Table 4.1, the final step represents an overall fold purification of 125.

相对纯化倍数
这个不是基础值，因为它可以通过上述的测定值计算得到，但经常是有用的。用每一步的纯度与第一步的纯度相比就可以得到。例如，在表4.1中，最终步骤的总纯化倍数是125。

有了前面的铺垫，这个纯化倍数值也就是给外行看看了。像在表中，起始物料0.8%的SDS-PAGE扫描纯度实际上是非常不准确的，由此而得到的纯化倍数125自然也是唬人的，用在发文章，申请经费时可以放个PPT给别人吹嘘吧。

2. The Importance of Footnotes
Every protein and purification is different and footnotes are needed to help the reader understand what has been done. There should be a footnote that indicates the amount of raw material used in the preparation being summarized. For recombinant protein expressed in bacteria, for example, one should always give the number of grams of wet weight cell pellet used in the preparation. It is also useful to know the volume of the bacterial culture used, but that in itself is not enough since, depending on the growth media and conditions, the yield of wet weight cell pellet can range from 1 to 80 g/L. Another useful footnote indicates how the protein amount was determined (e.g., sometimes a Bradford assay is used on the early steps, but absorbance and extinction coefficient is used on the final product).

脚注的重要性
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文中所说的要记录纯化所用的湿菌体量和发酵培养体积，非常赞同！我经常用的一个数据就是：mg蛋白/g湿菌体，就是1g湿菌体大肠杆菌可以纯化得到多少mg的目标蛋白。这个数据比纯化倍数什么的要有用得多，真实的反映了纯化过程的收率情况，换而言之，就是生产成本的情况。如果这个数据变动大了，肯定是发酵培养或纯化过程有问题出现。
由 （湿菌体/发酵体积） 可以看出来本批表达培养情况如何，这对纯化来说也是息息相关的。
蛋白分析方法要记录是不用说的了。

3. The Value of an SDS–Polyacrylamide Gel
Analysis on Main Protein Fractions I find that an SDS–polyacrylamide gel image is a very valuable complement to the purification summary table. If the same samples that represent the various steps in the purification table are also shown in a gel photo, then it is particularly easy to see the progress of the various fractionation steps toward production of a purified final target protein. The most useful gels are ones on which an equal proportion of the material at each step is loaded on the gel lanes.

SDS-PAGE电泳胶的价值
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同意。SDS-PAGE是我们做蛋白质纯化过程中最为亲密的伙伴。直观的看看每一步纯化的电泳图，很清晰地就表明了每一步纯化的情况怎么样。例如，目标蛋白的纯度，杂蛋白的去除，特别是个别杂蛋白条带的去除情况，这对组合纯化手段很有用。
另外，建议，如果不是为了发表文章，或者拿试验结果给老板看，电泳上样量建议大一些的好，不是为了看目标蛋白，而是为了看杂蛋白如何。同理，电泳样品buffer应该配制5*loading buffer的。

4. Some Common Mistakes and Problems
1. Use too many significant figures.
This is one of my pet peeves. I suspect that the concept of significant figures is no longer taught, because I find that a good 75% of the purification papers I review give protein amounts like 235.052 mg and yields like 46.72%. Just because a calculator or computer can divide two numbers and give one the result to eight figures does not mean that value is what one should enter into the table. Just remember that most protein quantification methods or enzyme assays are not accurate to better than 5–10%. When one writes 23.47 mg, one implies that it is not 23.46 or 23.48, but 23.47. In other words, one is implying that it is accurate to one part in over 2000 when it is not even clear that it is accurate to one part in 20 (is it 22, 23, or 24?). No numbers should be given to more than three significant figures and in general most percentages can be given to two significant figures. Also, remember that any number resulting from the division of two numbers each accurate to _10% will only be accurate to _20%.

一些常犯的错误和问题
用了太多的有效数字
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这一段有意义，应该看看。有效数字不是越多越好，也不代表越多越精确。由于蛋白质定量方法的误差大多数都是大于5-10%的，所以有效数字保持3位就足够了，对百分比的数值保持2位有效数字就够了。

2. Calculate values erroneously.
Remarkably many tables contain simple arithmetic errors. All numbers should be checked and rechecked before submission of a manuscript.

计算错误
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3. Use step yields instead of overall yields.
A step yield is the yield from a single step in the purification procedure, that is, the amount of target protein or activity after that step compared to that in the previous step of the procedure. A series of four fractionation steps might all give 60% step yields, but the overall yield is (0.6)4¼0.6_0.6_0.6_0.6¼0.13 or 13%. Overall yield is more useful. A procedure that gives an overall yield of a few percent may be due to a very lengthy and difficult purification of a rare or unstable protein, but more likely it indicates that the procedure has not been optimized very well.

应用分步收率代替总收率
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这一段隐含讲了一个原则性的问题。对一个蛋白质纯化过程来说，一定是步骤越短越好！即使你的每分步收率都达到60%，4步纯化过后你的纯化总收率也就是13%。
文中反对分步收率，对此有保留意见。诚然，总收率更有用，但分步收率也不是没有用的。如果某步的分步收率偏低，可以考虑对此步进行进一步的优化或改用其它方法。也就是所谓的限制性步骤吧，是我们重点突破的方向。

4. Calculate yield as yield of total protein.
Often I see a table in which the yield given is yield of total protein, rather than target protein. This is relatively useless information. Yield is always recovery of total target protein or activity.

用总蛋白的收率来表示收率
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一句话，收率只是目标蛋白量或活性的收率。用总蛋白来表现收率没有意义。