Cryo and detergents 晶体的冷冻和清洁剂

Cryo and detergents 晶体的冷冻和清洁剂

(Be aware of the potential for) detergent concentration mismatch between your mother liquor and the cryosolution. This particularly happens with vapor diffusion setups: there is a delicate balance of “free” detergent in the mother liquor versus the proportion of the detergent which is bound to the protein. Dropping a xtal into the cryosolution shocks the crystal with a bolus of extra free detergent. Hence, and counterintuitively, you may need to reduce the detergent concentration in the cryosolution to keep everything in balance. Try titrating down from 1% to even as low as 0.4% in the cryosolution. Under the conditions you are using the CMC of bOG is suppressed below the usual 0.67% (w/v).

Also, the behavior of many of the alkyl glycoside detergents is very temperature sensitive. So be careful about the temperature of all the solutions you use.

R. Michael Garavito, Ph.D.
Submitted to CCP4 bulletin board February 2007
Edited by Hampton Research Corp.

(请注意)母液和冷冻溶液之间的洗涤剂浓度不匹配的可能性。 这尤其发生在蒸汽扩散装置中:母液中的“游离”去污剂与与蛋白质结合的去污剂比例之间存在微妙的平衡。 将 xtal 滴入冷冻溶液中会用一团额外的游离洗涤剂冲击晶体。 因此,与直觉相反,您可能需要降低冷冻溶液中的洗涤剂浓度以保持一切平衡。 尝试在冷冻溶液中从 1% 滴定到甚至低至 0.4%。 在您使用的条件下,bOG 的 CMC 被抑制在通常的 0.67% (w/v) 以下。

此外,许多烷基糖苷去污剂的行为对温度非常敏感。 因此,请注意您使用的所有溶液的温度。

冷冻具体问题与结晶方案

冷冻具体问题与结晶方案

Cryo trouble?

Cryo Trouble? Give the following tips a try next time your crystal is fussy about freezing.

Try X-ray data collection at room temperature.

Evaluate other cryoprotectants. Try CryoPro from Hampton Research, which contains 36 unique cryopreservation reagents.
For more information visit http://hamptonresearch.com/product_detail.aspx?cid=30&sid=189&pid=30

Mixing of different cryoprotectants can have a superior protective effect over single component cryoprotectants of the same total concentration.

冷冻麻烦? 下次当您的水晶对冷冻很挑剔时,请尝试以下提示。

尝试在室温下收集 X 射线数据。

评估其他冷冻保护剂。 试试 Hampton Research 的 CryoPro,它包含 36 种独特的冷冻保存试剂。
欲了解更多信息,请访问 http://hamptonresearch.com/product_detail.aspx?cid=30&sid=189&pid=30

不同冷冻保护剂的混合比相同总浓度的单组分冷冻保护剂具有更好的保护效果。

Change the rate of cooling.  改变冷却速度。

a) Accelerate the rate of cooling. The fastest cooling rates have been achieved by blowing off the gas layer on liquid nitrogen during plunge cooling. (Hyperquenching for protein cryocrystallography, M. Warkentin et al, J. Appl. Cryst. (2006). 39, 805-811)

b) Slow the rate of cooling. Perhaps better suited to crystals with smaller solvent channels. The key to successful slow cooling of protein crystals is to carefully and completely remove all of the solvent from the surface of the crystal using oil such as Paratone-N, Perfluoropolyether, Mineral, Silicon, NVH or other. Remove ALL of the liquid from the surface of the crystals when using oil. (Slow cooling and temperature-controlled protein crystallography, Warkentin et al 10.1007/s10969-009-9074-y and Slow cooling of protein crystals, Warkentin et al, Volume 42, Part 5, Pages 944-952, October 2009) For more information visit http://hamptonresearch.com/menus.aspx?id=3&sid=138

a) 加快冷却速度。 通过在骤冷期间吹掉液氮上的气体层,可以实现最快的冷却速度。 (蛋白质低温晶体学的超淬灭,M. Warkentin 等人,J. Appl. Cryst. (2006). 39, 805-811)

b) 减慢冷却速度。 也许更适合溶剂通道较小的晶体。 成功缓慢冷却蛋白质晶体的关键是使用 Paratone-N、全氟聚醚、矿物、硅、NVH 或其他油等仔细彻底地去除晶体表面的所有溶剂。 使用油时,除去晶体表面的所有液体。 (缓慢冷却和温度控制的蛋白质晶体学,Warkentin 等人 10.1007/s10969-009-9074-y 和蛋白质晶体的缓慢冷却,Warkentin 等人,第 42 卷,第 5 部分,第 944-952 页,2009 年 10 月) 访问 http://hamptonresearch.com/menus.aspx?id=3&sid=138

Using salt as a crystallization reagent? Many salts are cryosalts, including malonate, formate, citrate, tartrate, acetate, Tacsimate and other organic acids, ammonium sulfate (>3.5 M), lithium sulfate, lithium chloride and other alkylammonium salts. For cryo try increasing your salt concentration by 20%. (Cryosalts: suppression of ice formation in macromolecular crystallography, K. A. Rubinson et al, Acta Cryst. (2000). D56, 996-1001 doi:10.1107/S0907444900007587.

Try the identical cryo procedure again with another crystal.

Vary the time and temperature of the crystal handling steps.

Check the liquid nitrogen level in your dewar and maintain a consistent level, day to day, week to week, month to month, year to year.

Try annealing. (Macromolecular crystal annealing: Techniques and cases studies. Bunick et al, The Rigaku Journal Vol. 15/ number 2/ 1998 and Macromolecular crystal annealing: overcoming increased mosaicity associated with cryocrystallography, Harp et al. (1998). Acta Cryst. D54, 622-8

使用盐作为结晶试剂?很多盐是冰晶盐,包括丙二酸盐、甲酸盐、柠檬酸盐、酒石酸盐、乙酸盐、Tacsimate和其他有机酸、硫酸铵(>3.5 M)、硫酸锂、氯化锂和其他烷基铵盐。对于冷冻,尝试将盐浓度提高 20%。 (冷冻盐:大分子晶体学中冰形成的抑制,K. A. Rubinson 等人,Acta Cryst. (2000). D56, 996-1001 doi:10.1107/S0907444900007587。

用另一个晶体再次尝试相同的冷冻程序。

改变晶体处理步骤的时间和温度。

检查杜瓦瓶中的液氮水平,并每天、每周、每月、每年保持一致的水平。

尝试退火。 (高分子晶体退火:技术和案例研究。Bunick 等人,理学杂志第 15 卷/第 2 期/1998 年和高分子晶体退火:克服与低温晶体学相关的镶嵌性增加,Harp 等人(1998 年)。Acta Cryst. D54, 622-8

Match the osmotic pressure of your cryoprotectant to the osmotic pressure of the reagent producing the crystal. Crystallization reagents with lower salt concentrations require a higher percentage of cryoprotectant for cryo protection than crystallization reagents with higher salt solutions (Cool data: quantity and quality. Elspeth Garman. Acta Cryst. (1999). D55, 1641-1653.). Osmolality tables (Weast, R. C. (1988-1989). Editor. Handbook of Chemistry; Physics, 69th ed. Boca Raton, Florida: CRC Press) can be used to estimate the osmolality of reagents. Another trick is to slowly concentrate a drop of the mother liquor by leaving the drop open to air and allowing the drop to slowly dry down, checking the mother liquor for clear glass freeze every few minutes. If you do not have X-rays to check for clear glass freeze, you can guesstimate by carefully placing the dewar under a dissecting microscope with overhead lighting. Focus on the surface of the liquid nitrogen, and bring the cooled loop into view just above the surface of the liquid nitrogen, where it is cold enough for guesstimate freezing.

使冷冻保护剂的渗透压与产生晶体的试剂的渗透压相匹配。与具有较高盐溶液的结晶试剂相比,具有较低盐浓度的结晶试剂需要更高百分比的冷冻保护剂来进行冷冻保护(酷数据:数量和质量。Elspeth Garman. Acta Cryst. (1999). D55, 1641-1653.)。渗透压表 (West, R. C. (1988-1989). Editor. Handbook of Chemistry; Physics, 69th ed. Boca Raton, Florida: CRC Press) 可用于估计试剂的渗透压。另一个技巧是慢慢浓缩一滴母液,方法是让一滴母液对空气敞开,让液滴慢慢干燥,每隔几分钟检查一次母液是否有透明玻璃结冰。如果您没有 X 射线来检查透明玻璃是否冻结,您可以通过将杜瓦瓶小心地放在带顶灯的解剖显微镜下进行猜测。专注于液氮的表面,将冷却回路置于液氮表面上方的视野中,那里足够冷,可以推测冻结。

Try high pressure cooling. (High-pressure cooling of protein crystals without cryoprotectants, Kim et al, Acta Cryst. (2005). D61, 881–890)

Cryoprotection of delicate crystals – Artem Evdokimov’s humble recipe.
http://www.xtals.org/crystal_cryo.pdf

Two words. Liquid propane.
http://cars9.uchicago.edu/biocars/pages/flashcooling.shtml

Roger S. Rowlett’s Cryoprotectant Strategy
1. Transfer crystals to mother liquor plus 30%v/v glycerol or ethylene glycol (sometimes lower depending on crystallization
reagent).
2. Transfer crystals to mother liquor plus 30%w/v glucose (or try sequential soaks in mother liquor plus 15%w/v and then 30%w/v glucose. Just a few seconds or minutes is usually enough). Glucose or other sugars often work when glycerol or ethylene glycol fails.
3. Try the “no-fail” in situ cryo method, which is a gradual buildup of cryoprotectant. This method is especially appropriate for crystals that cannot tolerate direct transfer to cryoprotectant solution, or for crystals that are especially sensitive to concentration changes in the mother liquor driven by drop evaporation. In our laboratory this method is routinely used with success on otherwise very sensitive crystals. This particular method is adapted for hanging drop crystallization. Ligands can be soaked in at the same time as cryopreservation if included in the cryoprotectant solution at 125% of the final, desired concentration.
a) Prepare a solution of artificial mother liquor plus 30% w/v glucose (40% v/v glycerol or another cryoprotectant can be substituted)
b) Remove a coverslip containing a drop with crystals to be cryoprotected and add 0.25 drop volume (DV) of cryoprotectant solutions (e.g. for a 4 uL drop add 1 uL of cryoprotectant solution). Replace coverslip on well and let stand for 5 minutes. Examine the crystals for cracking and/or dissolution.
c) Repeat the previous step with the following additional cryoprotectant additions: 0.25 DV, 0.50 DV, 1.00 DV, 2.00 DV. After each addition replace the coverslip over the well and let stand for 5 minutes. Examine crystals for cracking and/or dissolution.
d) After the last addition and 5 minute incubation, remove coverlip, fish out crystals with mounting loops and freeze directly in liquid nitrogen. The final glucose concentration will be 24%, sufficient to protect most crystallization solutions from ice formation upon freezing in liquid nitrogen.
This is very gentle, and often works when #1 and #2 does not, but in our hands nearly always increases mosaicity. (But mosaic is better than no diffraction.)
4. Try dragging crystals though Paratone-N to remove surface water from the crystal. This actually nearly always works for us, but is more fussy than #1 or #2, and it is easier to damage crystals during manipulation because of the viscosity of the oil.
I normally plunge protected crystals into liquid nitrogen after mounting.
Ice rings are a good indication of poor cryoprotection, but lack of diffraction could just be your crystals, too. For our latest dataset, we just sorted through 38 crystals until we found a good one. The key, as it turned out, is that all of our beautiful large crystals were apparently difficult to visualize disordered stacks of plates (we didn’t notice this until some fractured during cryo-soaks) whereas some of the small crystals were actually single crystals. We selected a decently diffracting small one and took loooooooooooong frames to get a good data set. (Roger S. Rowlett, Professor, Colgate University Presidential Scholar, Department of Chemistry, Colgate University, NY, USA)

Dig around in the cryoprotectant database for ideas. http://idb.exst.jaxa.jp/db_data/protein/search-e.php

Try 10% glycerol. (Jim Pflugrath)

Try 10 to 20% 2,3-butanediol. It can also reduce mosaicity. Try a quick dip, 30 to 60 seconds.

Fine tune the cryo concentration by screening 2.5% concentration increments, dipping a loop in the cryo buffer, freezing it and collecting X-Ray diffraction data to find the minimum concentration required that produces a clear glass and no ice rings.

Try dragging the crystal through a 1:1 mix of Paratone-N and Mineral oil until most or all the mother liquor from surrounding the crystal has been removed. (David Briggs)

Look back in your screen plates for a different crystal in different reagent, do not pass go, do not collect $200.00 and start over at the top of this tip.

Roger S. Rowlett 的冷冻保护剂策略
1. 将晶体转移到母液中,加入 30%v/v 甘油或乙二醇(有时根据结晶情况降低
试剂)。
2. 将晶体转移到加 30%w/v 葡萄糖的母液中(或尝试在母液中依次浸泡加 15%w/v 和 30%w/v 葡萄糖。通常只需几秒钟或几分钟就足够了)。当甘油或乙二醇失效时,葡萄糖或其他糖通常会起作用。
3. 尝试“不失败”的原位冷冻方法,这是一种逐渐积累的冷冻保护剂。这种方法特别适用于不能直接转移到冷冻保护剂溶液中的晶体,或对液滴蒸发驱动的母液浓度变化特别敏感的晶体。在我们的实验室中,这种方法通常成功地用于其他非常敏感的晶体。这种特殊的方法适用于悬滴结晶。如果在冷冻保护剂溶液中以最终所需浓度的 125% 包含配体,则可以在冷冻保存的同时浸泡配体。
a) 制备人工母液加 30% w/v 葡萄糖的溶液(可以用 40% v/v 甘油或其他冷冻保护剂代替)
b) 取出含有一滴需要冷冻保护的晶体的盖玻片,加入 0.25 滴体积 (DV) 的冷冻保护剂溶液(例如,对于 4 uL 的液滴,加入 1 uL 的冷冻保护剂溶液)。盖好盖玻片,静置 5 分钟。检查晶体是否破裂和/或溶解。
c) 重复上一步,添加以下额外的冷冻保护剂:0.25 DV、0.50 DV、1.00 DV、2.00 DV。每次添加后,更换孔上的盖玻片并静置 5 分钟。检查晶体是否开裂和/或溶解。
d) 最后一次添加和孵育 5 分钟后,取下盖玻片,用固定环捞出晶体并直接在液氮中冷冻。最终的葡萄糖浓度为 24%,足以保护大多数结晶溶液在液氮中冷冻时不会结冰。
这是非常温和的,并且通常在 #1 和 #2 不起作用时起作用,但在我们手中几乎总是会增加镶嵌性。 (但马赛克总比没有衍射要好。)
4. 尝试通过 Paratone-N 拖动晶体以去除晶体中的表面水。这实际上几乎总是对我们有用,但比 #1 或 #2 更挑剔,并且由于油的粘度,在操作过程中更容易损坏晶体。
我通常在安装后将受保护的晶体放入液氮中。
冰环是低温保护不良的一个很好的迹象,但缺乏衍射也可能只是你的晶体。对于我们最新的数据集,我们只是对 38 个晶体进行分类,直到找到一个好的晶体。事实证明,关键是我们所有美丽的大晶体显然很难看到无序的叠层板(直到在低温浸泡过程中有些破裂我们才注意到这一点),而一些小晶体实际上是单晶.我们选择了一个像样的衍射小帧,并用了 looooooooooooong 帧来获得一个好的数据集。 (Roger S. Rowlett,美国纽约州科尔盖特大学化学系科尔盖特大学校长学者教授)

在冷冻保护剂数据库中四处寻找想法。 http://idb.exst.jaxa.jp/db_data/protein/search-e.php

试试 10% 的甘油。 (吉姆·普弗拉格拉斯)

尝试使用 10 至 20% 的 2,3-丁二醇。它还可以减少镶嵌性。尝试快速浸泡 30 到 60 秒。

通过筛选 2.5% 的浓度增量来微调低温浓度,在低温缓冲液中浸一个循环,将其冷冻并收集 X 射线衍射数据,以找到产生透明玻璃和无冰环所需的最低浓度。

尝试将晶体拖过 Paratone-N 和矿物油的 1:1 混合物,直到去除晶体周围的大部分或全部母液。 (大卫布里格斯)

在您的筛板中查看不同试剂中的不同晶体,不要错过,不要收取 200.00 美元,然后从本提示的顶部重新开始。

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

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

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)

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.

 

Cryo buffer 冷冻缓冲液

Cryo buffer 冷冻缓冲液

We’ve had good success using the well solution directly as the foundation of a cryobuffer in several situations where crystals cannot be grown directly in the presence of cryoprotectant, and where crystals don’t tolerate transfer to artificial mother liquors. The basic protocol is as follows:

1. Remove 100 microliters of the well solution after crystals have grown

2. Split this sample into two 50 microliter aliquots.

3. Add 7.5 mg of dextrose (glucose) to the first aliquot and 15 mg of dextrose to the second. Dissolve by gentle pipeting with a wide-bore tip. This will give two sequential well solutions that now contain 15% and 30% w/v dextrose. If all the dextrose won’t go into the second aliquot, spin hard and remove the supernatant.

4. Transfer the crystal to aliquot 1, equilibrate for 3 minutes, then to aliquot number 2, then freeze. We’ve had a few crystals that routinely crack or blow up when transfered to artificial mother liquor that behave well when transfered to well solution plus glucose. We assume that there is some aspect of the crystal drop (pH, ionic tension, precipitant concentration) that is more effectively reproduced within the well than by separately prepared mother liquors. The nice thing about the protocol above is that you don’t get much of a volume increase when dry dextrose is dissolved in the well solution, so the components in the solution are not diluted.

Finally, if you don’t get a really good freeze, you can try to add about 5% v/v glycerol to aliquot 2 in addition to the 30% w/v dextrose. Reference: Personal communication from Barry Stoddard, Fred Hutchinson Cancer Research Center

冷冻缓冲液
在几种情况下,在存在冷冻保护剂的情况下晶体不能直接生长以及晶体不能转移到人工母液中的情况下,我们直接使用井溶液作为冷冻缓冲液的基础取得了很好的成功。基本协议如下:

1. 晶体生长后取出 100 微升孔溶液

2. 将此样品分成两份 50 微升的等份。

3. 在第一个等分试样中加入 7.5 mg 葡萄糖(葡萄糖),在第二个等分试样中加入 15 mg 葡萄糖。用大口径吸头轻轻吹打溶解。这将给出两个顺序井解决方案,现在包含 15% 和 30% w/v 葡萄糖。如果所有的葡萄糖都不会进入第二个等分试样,请用力旋转并去除上清液。

4. 将晶体转移到 1 号等分试样中,平衡 3 分钟,然后转移到 2 号等分试样中,然后冷冻。我们有一些晶体在转移到人造母液中时会经常破裂或爆炸,当转移到井溶液和葡萄糖中时表现良好。我们假设晶体液滴的某些方面(pH、离子张力、沉淀剂浓度)在井内比单独制备的母液更有效地再现。上述协议的好处是,当干葡萄糖溶解在孔溶液中时,体积不会增加太多,因此溶液中的成分不会被稀释。

最后,如果您没有得到很好的冷冻效果,除了 30% w/v 葡萄糖外,您可以尝试在等分试样 2 中添加约 5% v/v 甘油。参考:来自 Fred Hutchinson 癌症研究中心的 Barry Stoddard 的个人交流

A is for Aggregation 阻止包括蛋白质、肽和核酸在内的生物大分子的结晶

A is for Aggregation  A 代表聚合

Aggregation can be a deterrent to the crystallization of biological macromolecules including proteins, peptides, and nucleic acids. The presence of sample aggregation can be detected by either dynamic light scattering or native gel electrophoresis. Aggregation might be caused by hydrophobic patches on the surface of the sample, differently charged isoforms, differently phosphorylated isoforms, mixtures of methylated and non-methylated samples, glycosylation, as well as electrostatic interactions. Aggregation can be due to autologous aggregation where the protein is aggregating with itself or heterologous contamination where the sample is aggregating with other proteins. In the case of heterologous contamination, further purification of the sample should be seriously considered. In the case of autologous aggregation that precludes crystallization one might consider:

Using molecular biology to manipulate intra and inter molecule interactions by modifying the sample sequence (alter, add, or delete residues).

Use chemical additives to manipulate sample-sample and sample solvent interactions.
Detergents
Chaotropes (urea, guanidine hydrochloride, hydrochloric acid, etc)
Electrostatic agents
Alcohols (isopropanol, methanol, ethanol, etc)
Salts (sodium chloride, potassium chloride, sodium fluoride, etc)
Polyols (glycerol, PEG 400, etc)
Ligands, inhibitors, co-factors, and metals
Use temperature to prevent aggregation (0°C and 60°C)
Consider a fusion protein
Remove C-terminus or N-terminus
Truncate domains
Remove His-tag

In some cases aggregates can be removed by centrifugation or filtration.

In some cases the aggregates can be removed by mixing the sample with the crystallization reagent, allowing the sample to incubate for 15 minutes, centrifuging the sample/reagent mixture, removing the precipitate and setting the drop with the supernatant.

聚集可以阻止包括蛋白质、肽和核酸在内的生物大分子的结晶。可以通过动态光散射或天然凝胶电泳检测样品聚集的存在。聚集可能是由样品表面的疏水斑块、不同电荷的异构体、不同的磷酸化异构体、甲基化和非甲基化样本的混合物、糖基化以及静电相互作用引起的。聚集可能是由于蛋白质与自身聚集的自体聚集或样品与其他蛋白质聚集的异源污染。在异源污染的情况下,应认真考虑样品的进一步纯化。在排除结晶的自体聚集的情况下,可以考虑:

使用分子生物学通过修改样品序列(改变、添加或删除残基)来操纵分子内和分子间的相互作用。

使用化学添加剂来控制样品-样品和样品溶剂的相互作用。
洗涤剂
离液剂(尿素、盐酸胍、盐酸等)
静电剂
醇类(异丙醇、甲醇、乙醇等)
盐类(氯化钠、氯化钾、氟化钠等)
多元醇(甘油、PEG 400 等)
配体、抑制剂、辅因子和金属
使用温度防止聚集(0°C 和 60°C)
考虑融合蛋白
移除 C 端或 N 端
截断域
删除 His 标签

在某些情况下,可以通过离心或过滤去除聚集体。

在某些情况下,可以通过将样品与结晶试剂混合、让样品孵育 15 分钟、离心样品/试剂混合物、去除沉淀物并用上清液放置液滴来去除聚集体。

Trouble with protein storage 蛋白质储存问题

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

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

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.