Hampton HR2-861 Santovac Cryo Oil 油基防冻剂

Hampton HR2-861 Santovac Cryo Oil 油基防冻剂

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 Optimize Reagents Optimize – Cryoprotectants Santovac Cryo Oil

APPLICATIONS

  • Oil based cryoprotectant

应用
油基防冻剂

FEATURES

  • Low viscosity
  • Low surface tension
  • Cryoprotectant for soluble and membrane protein crystals
  • Not miscible with most solvents
  • Non toxic, non penetrating cryoprotectant

特征
低粘度
低表面张力
可溶性和膜蛋白晶体的冷冻保护剂
不与大多数溶剂混溶
无毒、非渗透性冷冻保护剂

DESCRIPTION

Used during cryocrystallography to displace and reduce the amount of water (mother liquor, reagent) on the crystal after the crystal is mounted in a cryoloop. Coating the crystal with oil can minimize evaporation from the crystal and reduce exposure and slow diffusion of air (oxygen) to the crystal.

Santovac Cryo Oil offers low viscosity, incredibly low vapor pressure, low surface tension, excellent chemical inertness and excellent thermal stability.

Dip the mounted crystal into the oil to displace surface water. Or add a small amount of Santovac to the crystallization drop before mounting the crystal. After adding the Santovac, mount the crystal using a Mounted Cryoloop. Withdraw the mounted crystal from the drop and the Santovac will coat the mounted crystal. Cryogenically cool the mounted crystal.

Santovac (Santovac 5) is a five ring polyphenyl ether (PPE) with very low vapor pressure and low reactivity. The oil is highly resistant to oxidation. The only elements in the pure polyphenyl ether are oxygen, hydrogen and carbon.

The fluid is chemically stable, non-corrosive, safe and non-toxic at normal operating temperatures.

Santovac is not miscible with water, paraffin oil, silicon oil, alkanes, or Parabar 10312 (Paratone). Santovac is miscible with acetone. Shelf life 5 years.

Santovac® is a trademark of Santovac® Fluids.

HR2-861  Santovac Cryo Oil 1 mL

描述
在晶体安装在低温回路中后,在低温晶体学期间用于置换和减少晶体上的水(母液、试剂)量。用油涂抹晶体可以最大限度地减少晶体的蒸发并减少空气(氧气)向晶体的暴露和缓慢扩散。

Santovac Cryo Oil 具有低粘度、极低的蒸气压、低表面张力、出色的化学惰性和出色的热稳定性。

将安装的晶体浸入油中以置换表面水。或者在安装晶体之前将少量的 Santovac 添加到结晶液滴中。添加 Santovac 后,使用 Mounted Cryoloop 安装晶体。从水滴中取出已镶嵌的水晶,Santovac 将覆盖已镶嵌的水晶。低温冷却安装的晶体。

Santovac (Santovac 5) 是一种五环聚苯醚 (PPE),具有极低的蒸气压和低反应性。该油具有很强的抗氧化性。纯聚苯醚中唯一的元素是氧、氢和碳。

该流体在正常工作温度下化学性质稳定、无腐蚀性、安全无毒。

Santovac 与水、石蜡油、硅油、烷烃或 Parabar 10312 (Paratone) 不混溶。 Santovac 可与丙酮混溶。保质期5年。

Santovac® 是 Santovac® Fluids 的商标。

HR2-861 Santovac 冷冻油 1 毫升

RELATED ITEM(S) 相关项目
CryoPro

Hampton HR2-861 Santovac Cryo Oil 油基防冻剂

REFERENCES  参考文献
1. Structure of the ‘open’ form of Aspergillus nidulans 3-dehydroquinate synthase at 1.7 Å resolution from crystal grown following enzyme turnover. C.E. Nichols, A.R. Hawkins and D.K. Stammersa. Acta Crystallographic Section D, Volume 60, Part 5, Pages 971-973, May 2004.

2. Crystal Structure of the Caspase Activator Human Granzyme B, a Proteinase Highly Specific for an Asp-P1 Residue. E. Estebanez-Perpina et al. Biol. Chem., Vol. 381, pp. 1203-1214, December 2000.

蛋白晶体出现冰环解决方案

蛋白晶体出现冰环解决方案

Ice Rings 冰环

Got ice rings and want to get rid of them?

While the crystal is in the beam, carefully try the following.

Try annealing the crystal by blocking the stream for a few seconds, allowing the crystal to thaw, then unblock the stream to cool the crystal again. (Reference: New techniques in macromolecular cryocrystallography: macromolecular crystal annealing and cryogenic helium, B. Leif Hansona, Constance A. Schallb and Gerard J. Bunick, Journal of Structural Biology
Volume 142, Issue 1, April 2003, Pages 77-87)

Try washing the crystal with liquid nitrogen. Using a pipette, carefully aspirate liquid nitrogen into the pipette, then dispense several drops or even a gentle stream over the crystal and mount. A liquid nitrogen wash can also be used to remove creeping ice from caps, pins and loops.

有冰环并想摆脱它们?

当晶体在光束中时,请仔细尝试以下操作。

尝试通过阻塞流几秒钟来使晶体退火,让晶体解冻,然后解除阻塞流以再次冷却晶体。 (参考:高分子低温晶体学新技术:高分子晶体退火和低温氦,B. Leif Hansona,Constance A. Schallb 和 Gerard J. Bunick,Journal of Structural Biology
第 142 卷,第 1 期,2003 年 4 月,第 77-87 页)

尝试用液氮清洗晶体。 使用移液器小心地将液氮吸入移液器中,然后在晶体上滴几滴甚至是轻柔的液流并安装。 液氮清洗也可用于去除盖子、销钉和环上的爬行冰。

在普通结晶试剂添加硫酸铵和盐

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。

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。

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。

Crystal Screen as an additive screen 附加屏幕

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。

 

Hampton HR2-795 1.0 M BIS-TRIS propane pH 7.0

Hampton HR2-795 1.0 M BIS-TRIS propane pH 7.0

上海金畔生物代理Hampton research品牌蛋白结晶试剂耗材工具等,我们将竭诚为您服务,欢迎访问Hampton research官网或者咨询我们获取更多相关Hampton research品牌产品信息。

BIS-TRIS propane Buffer

APPLICATIONS  应用

  • Crystallization grade BIS-TRIS propane pH 7.0 for formulating screens or for optimization   结晶级 BIS-TRIS 丙烷 pH 7.0,用于配制筛网或优化

FEATURES   特征

  • Sterile filtered
  • Formulated in Type 1+ ultrapure water: 18.2 megaohm-cm resistivity at 25°C, < 5 ppb Total Organic Carbon, bacteria free (<1 Bacteria (CFU/ml)), pyrogen free (<0.03 Endotoxin (EU/ml)), RNase-free (< 0.01 ng/mL) and DNase-free (< 4 pg/µL)

无菌过滤
在 1+ 型超纯水中配制:25°C 时电阻率为 18.2 兆欧厘米,< 5 ppb 总有机碳,无细菌(<1 细菌(CFU/ml)),无热原(<0.03 内毒素(EU/ml)) , 无 RNase (< 0.01 ng/mL) 和无 DNase (< 4 pg/µL)

DESCRIPTION

BIS-TRIS propane

Synonyms: 1,3-Bis[tris(hydroxymethyl)methylamino]propane
C11H26N2O6
CH2[CH2NHC(CH2OH)3]2
Mr 282.34
CAS Number [64431-96-5]
EC Number 264-899-3
Beilstein Registry Number 1786109
MDL Number MFCD00004689
PubChem Substance ID 24891934
Purity ≥ 99.0%
pKa1 = 6.8 at 25°C
pKa2 = 9.0 at 25°C

HR2-795 is titrated to pH 7.0 using HCl
Measured Conductivity Range: 41.9 – 43.5 mS/cm at 25°C
Measured Refractive Index Range: 1.39110 – 1.39222 at 20°C

HR2-795        1.0 M BIS-TRIS propane pH 7.0         100 mL

RELATED ITEM(S) 相关产品

REFERENCES  参考文献

1. Two-fold symmetry of crystalline DNA-EcoRI endonuclease recognition complexes. Grable, J; Frederick, CA; Samudzi, C; Jen-Jacobson, L; Lesser, D; Greene, P; Boyer, HW; Itakura, K; Rosenberg, JM. J Biomol Struct Dyn 1, 1149- 1160, 1984.

2. Non-native Local Interactions in Protein Folding and Stability: Introducing a Helical Tendency in the AII beta-Sheet alpha-Spectrin SH3 Domain. Prieto, J; Wilmans, M; Jimenez, MA; Rico, M; Serrano, L. J Mol Biol 268, 760- 778, 1997.

Hampton 丙二酸钠 3.4 M Sodium malonate pH 4.0

Hampton 丙二酸钠 3.4 M Sodium malonate pH 4.0

Sodium malonate  丙二酸钠

APPLICATIONS  应用

  • Crystallization grade Sodium malonate for formulating screens or for optimization用于筛选或优化的结晶级丙二酸钠

FEATURES  特征

  • Sterile filtered solution
  • Formulated in Type 1+ ultrapure water: 18.2 megaohm-cm resistivity at 25°C, < 5 ppb Total Organic Carbon, bacteria free (<1 Bacteria (CFU/ml)), pyrogen free (<0.03 Endotoxin (EU/ml)), RNase-free (< 0.01 ng/mL) and DNase-free (< 4 pg/µL)
  • 无菌过滤液
  • 在1+超纯水中配制:在25°C下电阻率为18.2兆,总有机碳<5 ppb,无细菌(<1细菌(CFU/ml)),无热原(<0.03内毒素(EU/ml)),无RNase(<0.01 ng/mL)和无DNase(<4 pg/L)

DESCRIPTION

The Sodium malonate offered by Hampton Research is Malonic acid (CAS Number 141-82-2) titrated to pH 4.0-8.0 using Sodium hydroxide as described in the publication “A comparison of salts for the crystallization of macromolecules. Alexander McPherson. Protein Science (2001), 10:418-422.”. In this same publication, Sodium malonate was reported to be more successful than any other salt, resulting in the crystallization of 19 of the 23 macromolecules, almost twice as effective as the next most successful salt, which was a draw between sodium acetate, sodium tartrate, sodium formate, and ammonium sulfate. The high success rate of Sodium malonate in producing crystals was even more impressive when an overall unique success rate with individual macromolecules was considered.

The use of Sodium malonate in place of Ammonium sulfate has been reported in one instance to improve the reproducibility of crystals and improve resistance to physicochemical shocks. Sodium malonate also acted as a good cryoprotectant (Xing and Xu 2003).

One may combine the four different sodium malonate stocks to create a pH versus sodium malonate concentration grid for screening or optimization. Refer to the pH/Dilution Table for Sodium malonate.

Based on the structure of malonic acid titrated with sodium hydroxide, there are no chiral carbons so it is not characterized as having enantiomeric structures, such as D or L.

Synonyms: Propanedioic acid or Malonic acid
C3H4O4 (before titration with NaOH)
Mr 104.06 (before titration with NaOH)
CAS Number 141-82-2
EC Number 205-503-0
Beilstein Registry Number 1751370
Merck 14,5710
RTECS OO0175000
MDL Number MFCD00002707
Purity > 99.0%
pKa1 2.8
pKa2 5.7

描述

Hampton研究提供的丙二酸钠是苹果酸(化学文摘社编号141-82-2),使用氢氧化钠滴定至pH4.0-8.0,载于出版物“大分子结晶盐的比较”,Alexander McPherson,“蛋白质科学”(2001),10:418-422。在同一份出版物中,据报道,丙二酸钠比任何其他盐都更成功,导致23种大分子中的19种结晶,几乎是第二种最成功的盐的两倍,即乙酸钠、酒石酸钠、甲酸钠和硫酸铵之间的结晶。当考虑到单个大分子的整体独特成功率时,丙二酸钠在晶体生产中的高成功率更令人印象深刻。

有报道称,用丙二酸钠代替硫酸铵可提高晶体的重现性,提高其抗物理化学冲击的能力。丙二酸钠也是一种良好的冷冻保护剂(邢和旭,2003年)。

一种方法是将四种不同的丙二酸钠组合起来,形成一个pH值相对于丙二酸钠浓度的网格,用于筛选或优化。请参阅丙二酸钠的pH/稀释表。

根据氢氧化钠滴定丙二酸的结构,不存在手性碳,不具有对映体结构,如D或L。

同义词:丙二酸或丙二酸
C3H4O4(用氢氧化钠滴定前)
Mr104.06(用氢氧化钠滴定前)
核证机关编号141-82-2
欧共体号码205-503-0
伯尔斯坦登记处编号1751370
默克14,5710
RTECS OO 0175000
MDL号码MFCD 00002707
纯度>99.0%
PKA 1 2.8
PKa25.7

HR2-747 Measured Conductivity Range: 73.3 – 75.3 mS/cm at 25°C
HR2-747 Measured Refractive Index Range: 1.38365 – 1.38425 at 20°C

HR2-749 Measured Conductivity Range: 77.2 – 78.0 mS/cm at 25°C
HR2-749 Measured Refractive Index Range: 1.39264 – 1.39300 at 20°C

HR2-751 Measured Conductivity Range: 73.4 – 75.2 mS/cm at 25°C
HR2-751 Measured Refractive Index Range: 1.39938 – 1.39973 at 20°C

HR2-707 Measured Conductivity Range: 71.0 – 73.7 mS/cm at 25°C
HR2-707 Measured Refractive Index Range: 1.40090 – 1.40098 at 20°C

HR2-807 Measured Conductivity Range: 71.8 – 75.0 mS/cm at 25°C
HR2-807 Measured Refractive Index Range: 1.40106 – 1.40115 at 20°C

HR2-747测量电导率范围:25°C下73.3~75.3 ms/cm
HR2-747测量折射率范围:1.38365~1.38425℃,20°C

HR2-749在25°C下的电导率范围:77.2~78.0 ms/cm
HR2-749测量折射率范围:1.39264~1.39300℃,20°C

HR2-751测量电导率范围:25°C下73.4~75.2 ms/cm
HR2-751测量折射率范围:1.39938~1.39973℃,20°C

HR2-707测量电导率范围:25°C下71.0~73.7 ms/cm
HR2-707测量折射率范围:1.40090~1.40098℃,20°C

HR2-807测量电导率范围:25°C下71.8~75.0 ms/cm
HR2-807测量折射率范围:1.40106~1.40115℃,20°C

HR2-747 3.4 M Sodium malonate pH 4.0 200 mL
HR2-749 3.4 M Sodium malonate pH 5.0 200 mL
HR2-751 3.4 M Sodium malonate pH 6.0 200 mL
HR2-707 3.4 M Sodium malonate pH 7.0 200 mL
HR2-807 3.4 M Sodium malonate pH 8.0 200 mL

REFERENCES  参考文献

1. A comparison of salts for the crystallization of macromolecules. Alexander McPherson. Protein Science (2001), 10:418-422.

2. Crystallization of the PX domain of cytokine-independent survival kinase (CISK): improvement of crystal quality for X-ray diffraction with sodium malonate. Yi Xing and Wenqing Xu. Acta Cryst. (2003). D59, 1816-1818.

3. 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. Acta Cryst. (2003). D59, 2356-2358.

4. Purification, crystallization and preliminary crystallographic analysis of Est25: a ketoprofen-specific hormone-sensitive lipase. Kim et al Acta Cryst. (2007). F63, 579–581.

Hampton HR2-539 四水酒石酸钾钠缓冲液

Hampton 四水酒石酸钾钠缓冲液

Potassium sodium tartrate tetrahydrate  四水酒石酸钾钠

上海金畔生物代理Hampton research品牌蛋白结晶试剂耗材工具等,我们将竭诚为您服务,欢迎访问Hampton research官网或者咨询我们获取更多相关Hampton research品牌产品信息。

APPLICATIONS 应用

  • Crystallization grade Potassium sodium tartrate tetrahydrate for formulating screens or for optimization 四水酒石酸钾结晶级筛选或优化

FEATURES  特征

  •  Sterile filtered solution
  • Formulated in Type 1+ ultrapure water: 18.2 megaohm-cm resistivity at 25°C, < 5 ppb Total Organic Carbon, bacteria free (<1 Bacteria (CFU/ml)), pyrogen free (<0.03 Endotoxin (EU/ml)), RNase-free (< 0.01 ng/mL) and DNase-free (< 4 pg/µL)
  • 无菌过滤液
  • 在1+超纯水中配制:在25°C下电阻率为18.2兆,总有机碳<5 ppb,无细菌(<1细菌(CFU/ml)),无热原(<0.03内毒素(EU/ml)),无RNase(<0.01 ng/mL)和无DNase(<4 pg/L)

DESCRIPTION

Potassium sodium tartrate tetrahydrate

Synonyms: L(+)-Tartaric acid potassium sodium salt, or Rochelle salt, or Seignette salt
KOCOCH(OH)CH(OH)COONa • 4H2O
C4H4KNaO• 4H2O
Mr 282.22
CAS Number [6381-59-5]
EC Number 206-156-8
Beilstein Registry Number 6113568
MDL Number MFCD00150989
PubChem Substance ID 24881874
Purity ≥ 99.5%

Measured pH Range: 8.4 – 9.1 at 25°C
Measured Conductivity Range: 81.1 – 98.5 mS/cm at 25°C
Measured Refractive Index Range: 1.37544 – 1.37666 20°C

描述

四水酒石酸钾钠

同义词:l(+)-酒石酸钾钠盐,或罗谢尔盐,或塞格奈特盐
KOCOCH(OH)CH(OH)Coona·4H2O
C4H4KNaO·4H2O
Mr 282.22
核证机关编号[6381-59-5]
欧共体编号206-156-8
伯尔斯坦登记处编号6113568
MDL号码MFCD 00150989
PubChem物质ID 24881874
纯度≥99.5%

测量pH值范围:8.4-9.1℃,25°C
测量电导率范围:81.1-98.5ms/cm,25°C
测量折射率范围:1.37544-1.37666 20°C

Al ≤0.0005%
As ≤0.00001%
Ba ≤0.0005%
Bi ≤0.0005%
Ca ≤0.001%
Cd ≤0.0005%
Cl ≤0.0005%
Co ≤0.0005%
Cr ≤0.0005%
Cu ≤0.0005%
Fe ≤0.0005%
Li ≤0.0005%
Mg ≤0.0005%
Mn ≤0.0005%
Mo ≤0.0005%
N ≤0.002%
Ni ≤0.0005%
Pb ≤0.0005%
PO4 ≤0.001%
SO4 ≤0.005%
Sr ≤0.0005%
Zn ≤0.0005%

HR2-539 1.5 M Potassium sodium tartrate tetrahydrate 200 mL

 

REFERENCES 参考文献

1. Cloning, purification, crystallization and preliminary crystallographic analysis of acylphosphatase from Pyrococcus horikoshii OT3. K. Miyazono, N. Kudo and M. Tanokura. Acta Cryst. (2004). D60, 1135-1136.

2. Crystallization and preliminary X-ray analysis of a xyloglucan endotransglycosylase from Populus tremula x tremuloides. Johansson P, Denman S, Brumer H, Kallas AM, Henriksson H, Bergfors T, Teeri TT, Jones TA. Acta Crystallogr D Biol Crystallogr. 2003 Mar;59(Pt 3):535-7.