还原剂

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R代表还原剂（Reducing Agents）

还原剂是一类能使其他物质被还原或获得电子的物质。在使其他物质被还原（获得电子）的过程中，还原剂本身被氧化了，也就是说还原剂通过自身被氧化（失去电子）而使其他物质被还原。
二硫苏糖醇DTT，beta-巯基乙醇，TCEP等是巯基类还原剂。还原剂可以保护蛋白质上自由的巯基不被氧化，从而避免蛋白质的聚集或变性。在结晶实验中，一般使用的还原剂工作浓度是1 － 10 mM 。
beta-巯基乙醇是上述三种还原剂中还原能力最差的一个，因为它本身只有一个巯基。它的效果一般只能维持2－3天。因此，在实验中要每隔2－3天补加一次beta-巯基乙醇来维持它的作用。由于beta-巯基乙醇是挥发性的，因此，可以在池液中加入beta-巯基乙醇，利用它的挥发扩散到液滴中去。
二硫苏糖醇DTT有两个巯基基团，其作用能维持3－7天。DTT的挥发性不像beta-巯基乙醇那么强，所以使用时应该直接加入到悬滴中去。DTT还可以通过微透析的方法加入。
TCEP的还原能力比beta-巯基乙醇和DTT都强，它的作用可以维持2－3周。TCEP能使结晶用的缓冲液酸化。与DTT类似，TCEP需要直接加入到悬滴中，因此可以考虑微透析法。
beta-巯基乙醇，DTT，和TECP的作用机制可能不同。像其他添加剂一样，如果你发现一类物质对样品的稳定性和结晶有利，则可以去筛选这一类物质中最适合你的样品的一种。我们建议是，最好这三种还原剂都尝试一下，去发现最适合的。
有报道指出还原剂作为没有自由巯基的蛋白的添加剂的情况下长出了晶体的成功案例。
还原剂可以与金属离子结合，金属离子其被还原，而还原剂的作用被抑制。这就使得用重金属原子作同晶置换法遇到了麻烦。可以使用EDTA来避免还原剂被金属离子抑制。但是，如果金属离子是你的样品蛋白维持活性所必须的，就要小心使用EDTA了。
碱性条件下，beta-巯基乙醇和TECP更稳定，效果要好于DTT。酸性条件下，TCEP的稳定性也要好过DTT。
DTT可以使Ni被还原，因此在用Ni柱纯化His标签的蛋白时要避免使用DTT，而应该考虑beta-巯基乙醇和TECP。
半胱氨酸也是一种还原剂。在结晶实验中，半胱氨酸局限性很明显，那是由于它容易形成六角形的晶体。但是，半胱氨酸是一种有效的添加剂，能带来明显的效果，不过需要铭记它的局限性。
如果预期到蛋白会被氧化，从制备样品开始就要加入还原剂，并且在整个流程中要不断补加保证其效果。在悬滴法中，可以在池液里加入还原剂，利用还原剂的挥发扩散，保证悬滴中还原性的环境。
在确定还原剂和样品比例时，需要考虑样品上自由巯基的数目和样品浓度。自由巯基数目越多，样品浓度越高，还原剂的用量就越大。
含砷化合物与还原剂不能共用。二甲胂和二甲胂酸钠是结晶中常使用的含砷化合物，它们不能与还原剂共同使用。其他不能与还原剂一起使用的物质还包括：硝酸铵，过氧化氢，高氯酸钾，硝酸钠。在使用某种物质之前，应该先查阅Material Safety Data Sheet (MSDS) 。
附原文：

	Reducing agents are substances that cause other chemical species to be reduced or gain electrons. In order for reducing agents to cause the gaining of electrons on some other chemical species, they must undergo oxidation. Therefore reducing agents undergo oxidation when they do their job. Dithiothreitol (DTT), beta-mercaptoethanol (beta-me), and Tris(2-Carboxyethyl)-Phosphine Hydrochloride (TCEP HCl) are sulfhydryl protective reducing agents. Reducing agents are typically used to prevent the oxidation of free sulfhydryl residues (cysteines) in the protein. Such oxidation can lead to non-specific aggregation of the sample, sample heterogeneity, inactivity, or denaturation of the sample. In a typical crystallization experiment, reducing agents are used in the concentration range of 1 to 10 mM in the crystallization drop. Beta-me has one sulfhydryl group and is the weakest of the three reducing agents discussed here, lasting perhaps two to three days. The supply of beta-me should be replenished every two to three days in the crystallization experiment to maintain the effectiveness of the reducing agent. Since beta-me is volatile, it can be added to the reservoir of vapor diffusion experiments for diffusion into the crystallization drop. DTT has two sulfhydryl groups and lasts about three to seven days in a typical crystallization experiment. DTT is not a strong volatile like beta-me (although it does possess a strong odor) and should be added directly to the crystallization drop when possible. Another consideration when working with DTT is to use the microdialysis method for crystallization since DTT can be added to the dialysis solution to replenish the supply of reducing agent. TCEP HCl is stronger than both beta-me and DTT, lasting about 2 to 3 weeks in a typical crystallization experiment. TCEP hydrochloride can acidify the crystallization solution. Like DTT, TCEP HCl needs to be added directly to the crystallization drop, hence, microdialysis is a consideration. Beta-me, DTT, and TCEP HCl can behave differently. Like all additives, if you find a class of compounds has an effect on sample stability or crystallization, then screen a variety of compounds in that class to see which one is best for your application. What we are trying to suggest is that like all additives, one might consider evaluating all three reducing agents to see which one works best for your sample. There have been reports where reducing agents have been used as successful crystallization additives where there were no free sulfhydryls in the sample. Reducing agents can bind metals and trace metal compounds, inactivating the reducing agent and the metal. This can make heavy atom derivatization in the presence of reducing agents, difficult and frustrating. EDTA can be added to the crystallization experiment to avoid inactivation of the reducing agent by metals. Keep in mind that if your sample needs metals for activity or stability that EDTA will keep metals from your sample. When working at an alkaline pH, beta-me and TCEP HCl are more stable than DTT. TCEP HCl is more stable at acid pH than DTT. DTT reduces nickel ions and can cause problems when purifying His-tagged proteins. To avoid this complication, try beta-me or TCEP HCl as a reducing agent instead of DTT when purifying His-tagged proteins. L-cysteine is also a reducing agent. It’s usefulness in crystallization is limited since it likes to form. small hexagonal plate shaped crystals. L-cysteine can be a useful crystallization additive, but keep those pesky hexagonal plates in the back of your mind when working with L-cysteine. If oxidation of the sample is expected or anticipated, reducing agents should be present during the preparation of the protein (when possible) and should be included, added, or replenished during the final preparation of the sample for crystallization. In vapor diffusion experiments, the reducing agent can be included or added to the crystallization reagent in the reservoir to minimize or prevent dilution of the reducing agent in the sample drop. When deciding on the appropriate concentration of reducing agent for the sample, consider the number of free sulfhydryls in the sample as well as sample concentration. More free sulfhydryls and higher sample concentration mean that one should consider using higher concentrations of reducing agent. Arsenical compounds are not compatible with reducing agents. Cacodylic acid, or sodium cacodylate is an arsenical compound and popular crystallization buffer and is not compatible with reducing agents. Other compounds not compatible with reducing agents include ammonium nitrate, hydroperoxide, potassium perchlorate, sodium nitrate. Check the Material Safety Data Sheet (MSDS) for these reagents for specifics about the incompatibilities of these chemicals with reducing agents. http://redondoself.blogbus.com/logs/20089137.html

Reducing agents are substances that cause other chemical species to be reduced or gain electrons. In order for reducing agents to cause the gaining of electrons on some other chemical species, they must undergo oxidation. Therefore reducing agents undergo oxidation when they do their job.

Dithiothreitol (DTT), beta-mercaptoethanol (beta-me), and Tris(2-Carboxyethyl)-Phosphine Hydrochloride (TCEP HCl) are sulfhydryl protective reducing agents. Reducing agents are typically used to prevent the oxidation of free sulfhydryl residues (cysteines) in the protein. Such oxidation can lead to non-specific aggregation of the sample, sample heterogeneity, inactivity, or denaturation of the sample. In a typical crystallization experiment, reducing agents are used in the concentration range of 1 to 10 mM in the crystallization drop.
Beta-me has one sulfhydryl group and is the weakest of the three reducing agents discussed here, lasting perhaps two to three days. The supply of beta-me should be replenished every two to three days in the crystallization experiment to maintain the effectiveness of the reducing agent. Since beta-me is volatile, it can be added to the reservoir of vapor diffusion experiments for diffusion into the crystallization drop.

DTT has two sulfhydryl groups and lasts about three to seven days in a typical crystallization experiment. DTT is not a strong volatile like beta-me (although it does possess a strong odor) and should be added directly to the crystallization drop when possible. Another consideration when working with DTT is to use the microdialysis method for crystallization since DTT can be added to the dialysis solution to replenish the supply of reducing agent.
TCEP HCl is stronger than both beta-me and DTT, lasting about 2 to 3 weeks in a typical crystallization experiment. TCEP hydrochloride can acidify the crystallization solution. Like DTT, TCEP HCl needs to be added directly to the crystallization drop, hence, microdialysis is a consideration.

Beta-me, DTT, and TCEP HCl can behave differently. Like all additives, if you find a class of compounds has an effect on sample stability or crystallization, then screen a variety of compounds in that class to see which one is best for your application. What we are trying to suggest is that like all additives, one might consider evaluating all three reducing agents to see which one works best for your sample.

There have been reports where reducing agents have been used as successful crystallization additives where there were no free sulfhydryls in the sample.

Reducing agents can bind metals and trace metal compounds, inactivating the reducing agent and the metal. This can make heavy atom derivatization in the presence of reducing agents, difficult and frustrating. EDTA can be added to the crystallization experiment to avoid inactivation of the reducing agent by metals. Keep in mind that if your sample needs metals for activity or stability that EDTA will keep metals from your sample.

When working at an alkaline pH, beta-me and TCEP HCl are more stable than DTT. TCEP HCl is more stable at acid pH than DTT.

DTT reduces nickel ions and can cause problems when purifying His-tagged proteins. To avoid this complication, try beta-me or TCEP HCl as a reducing agent instead of DTT when purifying His-tagged proteins.

L-cysteine is also a reducing agent. It’s usefulness in crystallization is limited since it likes to form. small hexagonal plate shaped crystals. L-cysteine can be a useful crystallization additive, but keep those pesky hexagonal plates in the back of your mind when working with L-cysteine.

If oxidation of the sample is expected or anticipated, reducing agents should be present during the preparation of the protein (when possible) and should be included, added, or replenished during the final preparation of the sample for crystallization. In vapor diffusion experiments, the reducing agent can be included or added to the crystallization reagent in the reservoir to minimize or prevent dilution of the reducing agent in the sample drop.

When deciding on the appropriate concentration of reducing agent for the sample, consider the number of free sulfhydryls in the sample as well as sample concentration. More free sulfhydryls and higher sample concentration mean that one should consider using higher concentrations of reducing agent.

Arsenical compounds are not compatible with reducing agents. Cacodylic acid, or sodium cacodylate is an arsenical compound and popular crystallization buffer and is not compatible with reducing agents. Other compounds not compatible with reducing agents include ammonium nitrate, hydroperoxide, potassium perchlorate, sodium nitrate. Check the Material Safety Data Sheet (MSDS) for these reagents for specifics about the incompatibilities of these chemicals with reducing agents.