标签归档：Hampton蛋白结晶

硫代硫酸钠防止分子间二硫键 -促进晶体生长和避免形成不稳定和弱衍射晶体

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Sodium thiosulfate to prevent intermolecular disulfide bridges

The presence of thiosulfate in the protein solution was essential to promote crystal growth and to avoid the formation of unstable and weakly diffracting crystals(1); this is likely to be a consequence of the intrinsic capability of the reduced thiol group of the active-site cysteine to form disulfide bridges, leading to the destabilization of the protein native structure. Sulfane sulfur-donor compounds such as Na2S2O3 are likely to either keep the protein in the persulfurated form or to prevent intermolecular disulfide bridges leading to unfolding and aggregation

硫代硫酸钠防止分子间二硫键
蛋白质溶液中硫代硫酸盐的存在对于促进晶体生长和避免形成不稳定和弱衍射晶体至关重要 (1)；这可能是活性位点半胱氨酸的还原硫醇基团形成二硫键的内在能力的结果，导致蛋白质天然结构的不稳定。硫烷硫供体化合物（如 Na2S2O3）可能使蛋白质保持过硫化形式或防止分子间二硫键导致展开和聚集 。

(2). References 参考文献

1. Crystallization and preliminary crystallographic characterization of LmACR2, an arsenate/antimonate reductase from Leishmania major. D. Bisacchi, Y. Zhou, B. P. Rosen, R. Mukhopadhyay and D. Bordo. Acta Cryst. (2006). F62, 976-979. 2. Bordo, D., Forlani, F., Spallarossa, A., Colnaghi, R., Carpen, A., Bolognesi, M. & Pagani, S. (2001). Biol. Chem. 382, 1245–1252.

Benzylkonium chloride-膜蛋白和可溶性蛋白用阳离子表面活性剂

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Benzylkonium chloride-膜蛋白和可溶性蛋白用阳离子表面活性剂

Try benzylkonium chloride (Fluka 12060). This cationic surface active agent has been reported to be useful as a crystallization additive with membrane proteins and may be useful for soluble proteins. We’ve been using it in the drop between 1 and 3% w/v in water. Try a 10% w/v stock solution in water and dilute into the drop to 1-3%.

苄基氯铵
试试苄基氯铵 (Fluka 12060)。 据报道，这种阳离子表面活性剂可用作膜蛋白的结晶添加剂，并可用于可溶性蛋白。 我们一直在水中使用 1% 到 3% w/v 的浓度。 尝试在水中使用 10% w/v 的储备溶液，然后将其稀释至 1-3%。

Crystal Screen as an additive screen 附加屏幕

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Crystal Screen as an additive screen 附加屏幕

Although Hampton Research does offer a specifically formulated Additive Screen (HR2-428), here is a tip when one already has a sparse matrix screen or two laying about the lab. When screening additives try adding 50 microliters of each Crystal Screen reagent to 950 microliters the “best” crystallization conditions thus far in order to see if any of the reagents in Crystal Screen might serve as good additives. Crystal Screen 2 is an especially good kit for this technique since Crystal Screen 2 contains numerous divalent cations, Jeffamine® Reagent, and a few other “novel” agents. Jeffamine® is a registered trademark of the Huntsman Petrochemical Corporation

Reference 参考文献

Crystallization of foot-and-mouth disease virus 3C protease: surface mutagenesis and a novel crystal-optimization strategy. Acta Crystallogr D Biol Crystallogr. 2005 May;61(Pt 5):646-50. Epub 2005 Apr 20. Birtley JR1, Curry S.

水晶屏幕作为附加屏幕
尽管 Hampton Research 确实提供了一种专门配制的添加剂屏幕 (HR2-428)，但当一个人在实验室周围已经有一个或两个稀疏矩阵屏幕时，这里有一个提示。 当筛选添加剂时，尝试将 50 微升的每种 Crystal Screen 试剂添加到迄今为止“最佳”结晶条件的 950 微升中，以查看 Crystal Screen 中的任何试剂是否可以作为良好的添加剂。 Crystal Screen 2 是该技术特别好的套件，因为 Crystal Screen 2 包含大量二价阳离子、Jeffamine® 试剂和一些其他“新型”试剂。 Jeffamine® 是 Huntsman Petrochemical Corporation 的注册商标。

 

What additive to use  蛋白结晶用用什么添加剂

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What additive to use  蛋白结晶用用什么添加剂

When trying to decide which additive might be useful during crystallization, try the following. If one has crystals and wants to try using additives to improve the crystal size or quality go back to the initial crystallization screening plates. Review the plates, looking for conditions where no precipitate nor crystals were/are observed. Now, review the results, looking for a common reagent ingredient present where no precipitant or crystals are found. For example, one might find that all drops with isopropanol remained clear. One could then try adding isopropanol to the current crystallization condition to see if the isopropanol could improve the crystal size and/or quality. If one observes a difference in the crystal in the presence of isopropanol, then one might consider evaluating other additives in the class of alcohols such as ethanol, methanol, tert-butanol and others. If one has no crystals, but plenty of precipitate, phase separation and clear drops, follow the above analysis and try adding the common reagent ingredient found in clear drop to those drops which contained precipitate or phase separation. It is possible this agent could improve or at least manipulate sample solubility. The above tip was submitted from Jarmila Jancarik from the laboratory of Sung Ho Kim at the University of California Berkeley. Thank you Jaru! When using this approach it might be reasonable to discern between concentration independent and dependent precipitation when trying to decide which agent to pursue as an additive. Try evaluating the concentration independent agents first and then look at the other agents if sample quantity permits. For example, if one observe precipitate in 15 to 40% PEG but not in 5 to 10% PEG, it might simply be a concentration. However, if one observes no precipitate in 45 to 60% v/v MPD one could guess that MPD is a reasonable agent to evaluate as an additive.

当试图决定在结晶过程中哪种添加剂可能有用时，请尝试以下方法。如果有晶体并想尝试使用添加剂来改善晶体尺寸或质量，请返回最初的结晶筛选板。检查板，寻找没有观察到沉淀或晶体的条件。现在，查看结果，寻找没有发现沉淀剂或晶体的常见试剂成分。例如，人们可能会发现所有含有异丙醇的液滴都保持透明。然后可以尝试将异丙醇添加到当前的结晶条件中，以查看异丙醇是否可以改善晶体尺寸和/或质量。如果在异丙醇存在下观察到晶体的差异，那么可以考虑评估醇类中的其他添加剂，例如乙醇、甲醇、叔丁醇等。如果没有晶体，但有大量的沉淀、相分离和透明液滴，请按照上述分析，尝试将透明液滴中常见的试剂成分添加到含有沉淀或相分离的液滴中。该试剂有可能改善或至少控制样品溶解度。上述提示由加州大学伯克利分校 Sung Ho Kim 实验室的 Jarmila Jancarik 提交。谢谢雅鲁！当使用这种方法时，在尝试决定使用哪种试剂作为添加剂时，区分与浓度无关的沉淀和依赖沉淀可能是合理的。尝试先评估与浓度无关的试剂，然后在样本数量允许的情况下查看其他试剂。例如，如果观察到 15% 到 40% PEG 中的沉淀，但 5% 到 10% PEG 中没有，则它可能只是一种浓度。然而，如果在 45 至 60% v/v MPD 中没有观察到沉淀，则可以猜测 MPD 是一种可以作为添加剂进行评估的合理试剂。