标签归档：Hampton代理

在普通结晶试剂添加硫酸铵和盐

发表于由

Cryo for Ammonium sulfate and salt based crystallization reagents   用于硫酸铵和盐基结晶试剂的 Cryo

Increase the concentration of ammonium sulfate (cryosalt) (or primary salt acting as a precipitant) in increments of 10% in the presence of 5 to 10% v/v Glycerol.

Evaluate 25-30% w/v glucose, or trehalose or sucrose as cryoprotectants for crystals grown in Ammonium sulfate. Acta Cryst. (2002). D58, 1664-1669. Crystallization of RNA/protein complexes. M. Garber, G. Gongadze, V. Meshcheryakov, O. Nikonov, A. Nikulin, A. Perederina, W. Piendl, A. Serganov and S. Tishchenko.

15 to 30% v/v Ethylene glycol, DMSO, or Glycerol.

Try 1 – 2 M Sodium malonate as a cryoprotectant. Acta Cryst. (2003). D59, 2356-2358. Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals. T. Holyoak, T. D. Fenn, M. A. Wilson, A. G. Moulin, D. Ringe and G. A. Petsko.

Place a drop of 75% v/v Paratone-N, 25% v/v Paraffin Oil next to the drop containing the crystal. Remove the crystal from the drop using a CryoLoop and dip the crystal into the oil. Keeping the CryoLoop and crystal immersed, gently move the CryoLoop containing the crystal from the oil into the reagent, back into the oil, back into the reagent, repeating this several times to remove some of the aqueous (reagent) layer from the crystal surface. Cryo cool the crystal.

在 5 至 10% v/v 甘油存在的情况下，以 10% 的增量增加硫酸铵（冰晶盐）（或用作沉淀剂的初级盐）的浓度。

评估 25-30% w/v 葡萄糖、海藻糖或蔗糖作为在硫酸铵中生长的晶体的冷冻保护剂。晶体学报。 （2002 年）。 D58，1664-1669。 RNA/蛋白质复合物的结晶。 M. Garber、G. Gongadze、V. Meshcheryakov、O. Nikonov、A. Nikulin、A. Perederina、W. Piendl、A. Serganov 和 S. Tishchenko。

15 至 30% v/v 乙二醇、DMSO 或甘油。

尝试 1 – 2 M 丙二酸钠作为冷冻保护剂。晶体学报。 （2003 年）。 D59，2356-2358。丙二酸盐：一种用于盐生大分子晶体的多功能冷冻保护剂和稳定溶液。 T. Holyoak、T. D. Fenn、M. A. Wilson、A. G. Moulin、D. Ringe 和 G. A. Petsko。

在含有晶体的液滴旁边放置一滴 75% v/v Paratone-N、25% v/v 石蜡油。使用 CryoLoop 从液滴中取出晶体并将晶体浸入油中。保持 CryoLoop 和晶体浸没，轻轻地将含有晶体的 CryoLoop 从油中移入试剂中，再移回油中，再移回试剂中，重复几次以去除晶体表面的一些水（试剂）层。低温冷却晶体。

Cleaning a CryoLoop   CryoLoop环的清洗方法

发表于由

Cleaning a CryoLoop   CryoLoop环的清洗方法

Soak the CryoLoop in a 0.5% detergent solution in deionized water for 15 minutes. Gently rinse the CryoLoop in deionized water. Allow the CryoLoop to air dry.

If more rigorous cleaning is desired or one does not wish to risk the presence of trace amounts of residual detergent, soak the CryoLoop in 5.0 M Urea formulated in deionized water for 15 minutes. Gently rinse the CryoLoop in deionized water. Repeat three times. Allow the CryoLoop to air dry.

将 CryoLoop 浸泡在 0.5% 去离子水中的清洁剂溶液中 15 分钟。 在去离子水中轻轻冲洗 CryoLoop。 让 CryoLoop 风干。

如果需要更严格的清洁或不想冒残留洗涤剂残留的风险，请将 CryoLoop 浸泡在 5.0 M 去离子水中配制的尿素中 15 分钟。 在去离子水中轻轻冲洗 CryoLoop。 重复三遍。 让 CryoLoop 风干。

The potential benefits of cryogenic data collection 低温数据收集的潜在好处

发表于由

The potential benefits of cryogenic data collection 低温数据收集的潜在好处

Reduced radiation damage.

Decreased thermal motion and disorder.

Potential for improved resolution.

Increased crystal lifetime.

Crystals can be stored and shipped.

减少辐射伤害。

减少热运动和紊乱。

提高分辨率的潜力。

增加晶体寿命。

晶体可以储存和运输。

Mounting thin crystals 安装晶体环方法

发表于由

Mounting thin crystals 安装晶体环方法

To mount very thin crystals onto cryoloops, first dip the nylon loop into 0.5% Formvar solution (Fluka # 09819) to form a thin film. The film provides extra support for fragile crystals, and can result in much sharper reflections with just slightly higher background. To clean the loop, dip it in alcohol to dissolve the support. Two notes: (1) the technique works only for crystals grown without organic solvents, and (2) take precautions not to breathe vapor from the formvar solution–the solvent is 1,2 dichloroethane. Formvar is a standard support for electron microscopy grids.

要将非常薄的晶体安装到冷冻环上，首先将尼龙环浸入 0.5% Formvar 溶液 (Fluka # 09819) 中以形成薄膜。 该薄膜为易碎的晶体提供了额外的支撑，并且可以在背景稍高的情况下产生更清晰的反射。 要清洁环，请将其浸入酒精中以溶解支撑物。 两个注意事项：(1) 该技术仅适用于在没有有机溶剂的情况下生长的晶体，以及 (2) 采取预防措施，不要从 formvar 溶液中吸入蒸汽——溶剂是 1,2 二氯乙烷。 Formvar 是电子显微镜网格的标准支持。

PEGs for cryo 用于冷冻的 PEG（聚乙二醇）

发表于由

PEGs for cryo 用于冷冻的 PEG（聚乙二醇）

High molecular PEGS are also good cryoprotectants. If crystals are obtained from relatively high concentration of PEGS (e.g., 30% of PEG 3350), you can cryoprotect them simply by raising the concentration of the PEG a little bit (e.g., 40% of PEG 3350).

高分子PEGS也是很好的冷冻保护剂。 如果晶体是从相对高浓度的 PEGS（例如 30% 的 PEG 3350）中获得的，您可以通过稍微提高 PEG 的浓度（例如 40% 的 PEG 3350）来冷冻保护它们。

参考文献

J. Appl. Cryst. (2006). 39, 244-251
Effects of cryoprotectant concentration and cooling rate on vitrification of aqueous solutions. V. Berejnov, N. S. Husseini, O. A. Alsaied and R. E. Thorne
Synopsis: Critical concentrations required for vitrification of aqueous solutions are determined for fourteen common cryoprotectants, for sample volumes ranging over four orders of magnitude and covering the range of interest in protein crystallography.

 

Trouble with protein storage 蛋白质储存问题

发表于由

Trouble with protein storage 蛋白质储存问题

Try 3 to 5% 1,2-propanediol in the protein buffer as a substitute for glycerol to stabilize proteins. Annie Hassell, Glaxo Smithkline 2009

尝试在蛋白质缓冲液中加入 3% 到 5% 的 1,2-丙二醇作为甘油的替代品来稳定蛋白质。 安妮哈塞尔，葛兰素史克 2009

Dissolving hydrophobic additives into oil-用于静滴蒸汽扩散或油下微量

发表于由

Dissolving hydrophobic additives into oil-用于静滴蒸汽扩散或油下微量

Try dissolving the small molecule additive into paraffin or silicon oil, and use this mixture to cover the sample drop. This can be used with sitting drop vapor diffusion or with microbatch under oil. The oil acts as a reservoir that may contain excess small molecule that (you hope) will be fed into the crystals.

将疏水性添加剂溶解到油中
尝试将小分子添加剂溶解在石蜡或硅油中，然后用这种混合物覆盖样品液滴。 这可用于静滴蒸汽扩散或油下微量。 油充当储存器，可能含有过量的小分子（您希望）将被送入晶体中。

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

发表于由

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

发表于由

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

发表于由

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。