Reducing agents
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.
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.
TAG:
标题搜索
日历
|
|||||||||
| 日 | 一 | 二 | 三 | 四 | 五 | 六 | |||
| 1 | |||||||||
| 2 | 3 | 4 | 5 | 6 | 7 | 8 | |||
| 9 | 10 | 11 | 12 | 13 | 14 | 15 | |||
| 16 | 17 | 18 | 19 | 20 | 21 | 22 | |||
| 23 | 24 | 25 | 26 | 27 | 28 | 29 | |||
| 30 | |||||||||
我的存档
数据统计
- 访问量: 2524
- 日志数: 62
- 建立时间: 2011-08-27
- 更新时间: 2015-11-02
