这项工作是由宝来惠康研究者奖，以YM和布朗Coxe从美国耶鲁大学博士后奖学金到MD的支持。

材料： <ul><li>蛋白质样品 - 溶菌酶（50毫克/毫升） </li><li>悬滴24井托盘</li><li>坐滴24井托盘</li><li> Microbatch结晶96以及托盘</li><li>结晶解决方案（无论是商业用或自制） </li><li>硅脂</li><li> 5 mL注射器无鲁尔锁</li><li>硅化盖幻灯片</li><li>光封箱胶带</li><li>石蜡</li><li> 0.1-2微升微量低保留提示</li><li>镊子</li><li>专业湿巾</li></ul> 1。悬/坐在DROP PROCEDURE： <ol><li>对于一个初始画面，可以使用市售的屏幕，利用稀疏矩阵，不完整的试验条件的阶乘方法。稀疏矩阵的不完整的阶乘方法是有失偏颇和选择知名的大分子结晶条件（卡特和卡特，1979年; Cudney等，1994; Jancarik和Kim，1991; Jancarik等，1991） 。如果是已知的结晶条件，可以组装一个狭窄的屏幕，不同的沉淀剂的浓度和pH值周围的原始命中。最常用的沉淀剂是聚乙二醇（PEG），硫酸铵。总之，这两个沉淀结晶（吉利兰等，1994;伯等，2003;。Page等人 ，2003年）所使用的所有记录的大分子沉淀约60 ％的帐户。 </li><li>准备好您的结晶解决方案。通常情况下，这些条件相结合，从沉淀剂，盐某种设置pH值和一个缓冲。在某些情况下的添加剂可能会增加特别感兴趣的蛋白质。所有股票的解决方案应使用0.22微米过滤器过滤。原液的一些人可能会非常粘稠（50％聚乙二醇，甘油，PEG - MME等）。 <ul><li>准备1.5 mL的0.6，0.8，1.0，1.2，1.4，1.6 M氯化钠0.1 M NaOAc pH值4.0，4.3，4.6和4.9（共24解决方案） </li></ul></li><li>解冻蛋白质样品，并置于冰上。蛋白质股票应存放于-80 ° C，除非感兴趣的蛋白质是敏感的，冻结/解冻循环（若有，储存在4 ° C）。典型的蛋白质浓度范围在5-50毫克/毫升，具有较高的浓度通常提供更好的结果。 </li><li>旋转样品15min/18 000xg / 4 ° C - 一些蛋白质往往部分聚合和沉淀后解冻步骤。这些聚集体，可促进蛋白质分子的其余部分无定形沉淀，因此彻底的降速将确保你只在你的样品的可溶性分子。保存，直到成立托盘上冰蛋白。 </li><li>使用紫外分光光度计在280 nm处的吸光度确定蛋白浓度。吸光度读数和蛋白质的消光系数，可以计算出蛋白浓度。消光系数，可以计算出与ProtParam工具Expasy网站<a href="http://ca.expasy.org/tools/protparam.html">：</a> http://ca.expasy.org/tools/protparam.html </li><li> 24挂/ 500μL沉淀的解决方案，根据托盘设置方案（图2B）坐在下降托盘填写井。创建硅脂环周围以及边缘。环应该有一个小的差距，以防止空气井是密封的压力恢复。坐在滴，有没有油脂的需要，因为该井是用胶带密封（图2A）。 </li><li>以封面幻灯片和压缩空气喷雾，或专业擦拭干净 - 避免灰尘会缓解结晶下降的解释和消除污染。报考滴，不使用封面幻灯片。相反，以及包含其中的蛋白质和沉淀剂混合货架。 </li><li>将等量的蛋白质样品和水库的解决方案，封面上的幻灯片。作为优化过程的一部分，可能会尝试不同的卷的蛋白质和沉淀。使用更多的蛋白质比在蛋白质平衡后下降，稀蛋白质样品，是可取的和缓慢的晶体核或增长的净含量沉淀的结果。任何其他非易失性物质下降的解决方案也将集中由相同的因素。吹打到封面幻灯片的蛋白质和水库解决方案时，要非常小心，以避免泡沫的形成。这往往发生时空气吹吸管。 <ul><li>悬滴：0.02 M NaOAc pH值4.9负载为50毫克/毫升溶菌酶2μL，封面幻灯片的中心，以及加入2μL水库的解决方案。 </li><li>坐在下降：负载2μL50 mg / mL的溶菌酶溶液的货架的中心，以及加入2μL水库解决方案。 </li></ul></li><li>轻轻地翻转盖幻灯片，以及顶部的油脂环奠定下来。油脂轻轻向下按，使空气可以通过缺口从井逃脱公关事件的空气在井压力恢复和保持密封良好。以及在空气压力积聚可提高盖滑动，影响密封性好。在坐滴法，光学透明胶带是用来盖井。因此，在此方法密封将每行/列。 </li><li>可移动的未来以及，直到托盘完成。 </li><li>检查安装后的托盘 - 消除灰尘颗粒和其他污染物，可减少蛋白质晶体的积极识别假。 </li><li>使用评分表，记录自己的实验。 </li><li>托盘放置在所需的孵化温度，一般在4 ° C和室温。 20℃是最常用的成功。小心轻轻地来处理托盘，并避免任何晃动。要小心，而开幕式和闭幕式孵化器的门。振动或转让或孵化期间温度变化可以防止晶体生长的晶体质量产生负面影响。冲击吸收材料（如包装泡沫），可以用来作为填充下，水晶托盘。 </li><li>检查水晶托盘翌日，然后每隔几天，总是小心处理的托盘。文件中的任何一个托盘具体的评分表中的结果和下降形态。晶体通常需要2-5天出现，但晶体偶尔会出现更快（几乎立即）或更高（可达数月！）。一旦结晶条件已经确定，晶体的生长速率是已知的，最好是离开的托盘不受干扰，直到晶体的出现和成长至少他们的最终大小的一半。 ，托盘不受干扰，可以减少晶体的成核事件的数量，从而产生更大的晶体更小的数字。 </li></ol> 2。 Microbatch程序： <ol><li>蛋白质样品和沉淀剂制备如上所述。 </li><li>空气喷涂一个新的microbatch托盘，以避免灰尘和其他大颗粒。 </li><li>在microbatch托盘，填补石蜡油3毫米高（只够盖井）。卸下石油的访问。 </li><li>装入石油预填充以及1μL蛋白质溶液（50毫克/毫升溶菌酶） - 确保移液器的解决方案，直接到井底（图2A）。 </li><li>根据托盘设置方案（图2B）装入1μL沉淀的解决方案 - 确保下拉汇以及和保险丝的底部与蛋白质液滴。 </li><li>移动到下一个井，直到完成托盘。 </li><li>按照步骤悬/坐滴程序11-14。 </li></ol> 3。代表性的成果： 结晶通常被称为X射线晶体学的瓶颈。一个稀疏矩阵的不完整的沉淀条件的阶乘屏幕上通常会产生许多不同类型的蛋白质聚集和沉淀，其中大单晶。如果蛋白质或沉淀剂的浓度过高，人们可以看到褐色的问题，没有独特的形状和尺寸（无定形沉淀）。下降的解决方案是欠当，往往会被彻底清除，没有任何一种沉淀。图3显示了沉淀现象和晶体（德绍等人 ，2006年）的几个例子。更详细的解释更多的沉淀现象，可以<a href="http://xray.bmc.uu.se/terese/tutorials.html">发现</a>在http://xray.bmc.uu.se/terese/tutorials.html。 <img src="/files/ftp_upload/2285/2285fig1.jpg" alt="图1" /> 图1。蛋白质结晶的原则 。 蛋白质结晶的原则。在气相扩散实验（一）平等的沉淀和蛋白质的数量在下降。水漫出的沉淀和蛋白质浓度将增加一倍，直到下降，水库的解决方案之间实现平衡。 （二）在间歇结晶沉淀和蛋白浓度在实验过程中不改变。 A点 - 蛋白质停留欠无晶体才能形成，B点 - 蛋白质核发生，晶体开始形成和蛋白质在溶液中的浓度下降到饱和。 C点 - 蛋白质沉淀，但晶体可能继续增长。 （三）透析结晶用凝胶堵塞毛细血管。晶体出现盐扩散出的蛋白质样品，整个凝胶插件（和/或沉淀扩散的蛋白质样品）。 <img src="/files/ftp_upload/2285/2285fig2.jpg" alt="图2" /> 图2。典型的蛋白质结晶实验大纲 。 （一）悬滴气相扩散，坐滴气相扩散和microbatch蛋白质结晶过程。在每一种情况下，一个浓缩的蛋白质样品的体积小的precipitan相同或更小的体积混合T解决方案，并允许以平衡。 （二）设置鸡溶菌酶，24孔托盘的结晶，是与不同浓度的氯化钠（沉淀）和0.1 M为在不同pH值的缓冲醋酸钠（4.0 - 4.9）。 <img src="/files/ftp_upload/2285/2285fig3.jpg" alt="图3" /> 图3。蛋白质结晶实验的典型结果 。 （一）无定形沉淀。当蛋白质或沉淀（或两者）在高浓度。 （二）相分离。蛋白质或清洁剂可能分开到不同阶段时，在高浓度的某些沉淀混合。 （三）杆形蛋白质晶体的AtCSN7获得聚乙二醇8000和醋酸镁（德绍等，2006）。 （四）在1.0 M氯化钠和醋酸钠的pH值4.9溶菌酶晶体获得。 （e）根据饱和滴通常保持清醒。摄影卡察夫德绍。

<ol>
	<li>Bergfors, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein crystallization : techniques, strategies, and tips : a laboratory manual. , (1999).</li><li>Carter, C. W., Carter, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+crystallization+using+incomplete+factorial+experiments.">Protein crystallization using incomplete factorial experiments.</a> J Biol Chem. 254, 12219-12223 (1979).</li><li>Cudney, R., Patel, S., Weisgraber, K., Newhouse, Y., McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+optimization+strategies+for+macromolecular+crystal+growth.">Screening and optimization strategies for macromolecular crystal growth.</a> Acta Crystallogr D Biol Crystallogr. 50, 414-423 (1994).</li><li>Dessau, M., Chamovitz, D. A., Hirsch, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+purification+and+crystallization+of+a+PCI+domain+from+the+COP9+signalosome+subunit+7+(CSN7).">Expression, purification and crystallization of a PCI domain from the COP9 signalosome subunit 7 (CSN7).</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 62, 1138-1140 (2006).</li><li>Gilliland, G. L., Tung, M., Blakeslee, D. M., Ladner, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Macromolecule+Crystallization+Database,+Version+3.0:+new+features,+data+and+the+NASA+archive+for+protein+crystal+growth+data.">Biological Macromolecule Crystallization Database, Version 3.0: new features, data and the NASA archive for protein crystal growth data.</a> Acta Crystallogr D Biol Crystallogr. 50, 408-413 (1994).</li><li>Jancarik, J., Kim, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparse+matrix+sampling:+a+screening+method+for+crystallization+of+proteins.">Sparse matrix sampling: a screening method for crystallization of proteins.</a> J Appl Cryst. 23, 409-411 (1991).</li><li>Jancarik, J., Scott, W. G., Milligan, D. L., Koshland, D. E., Kim, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+and+preliminary+X-ray+diffraction+study+of+the+ligand-binding+domain+of+the+bacterial+chemotaxis-mediating+aspartate+receptor+of+Salmonella+typhimurium.">Crystallization and preliminary X-ray diffraction study of the ligand-binding domain of the bacterial chemotaxis-mediating aspartate receptor of Salmonella typhimurium.</a> J Mol Biol. 221, 31-34 (1991).</li><li>Kimber, M. S., Vallee, F., Houston, S., Necakov, A., Skarina, T., Evdokimova, E., Beasley, S., Christendat, D., Savchenko, A., Arrowsmith, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+mining+crystallization+databases:+knowledge-based+approaches+to+optimize+protein+crystal+screens.">Data mining crystallization databases: knowledge-based approaches to optimize protein crystal screens.</a> Proteins. 51, 562-568 (2003).</li><li>Lorber, B., Sauter, C., Theobald-Dietrich, A., Moreno, A., Schellenberger, P., Robert, M. C., Capelle, B., Sanglier, S., Potier, N., Giege, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+growth+of+proteins,+nucleic+acids,+and+viruses+in+gels.">Crystal growth of proteins, nucleic acids, and viruses in gels.</a> Prog Biophys Mol Biol. 101, 13-25 (2009).</li><li>McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Crystallization of biological macromolecules. , (1999).</li><li>McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+protein+crystallization.">Introduction to protein crystallization.</a> Methods. 34, 254-265 (2004).</li><li>Page, R., Grzechnik, S. K., Canaves, J. M., Spraggon, G., Kreusch, A., Kuhn, P., Stevens, R. C., Lesley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+crystallization+strategy+for+structural+genomics:+an+optimized+two-tiered+crystallization+screen+against+the+Thermotoga+maritima+proteome.">Shotgun crystallization strategy for structural genomics: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome.</a> Acta Crystallogr D Biol Crystallogr. 59, 1028-1037 (2003).</li></ol>

在这篇文章中我们将介绍和演示蛋白质结晶当前的协议。自一个多步骤的过程中有几个注意事项要注意的一个需要。当工作与极少量（0.5-2μL）下拉由于蒸发干燥，是一个主要问题。因此，建议工作在良好的控制环境低的空气流动性，高湿度和严格的温度控制，并采取了一种技术，最大限度地减少暴露露天下降。出于这个原因，它运行在一个组织良好的的工作环境是必不可少的。所有试剂应准备?...

<table border="1">
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<tr>
<td>New Item</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>L6876-1G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>24 well VDX Plate</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-142</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>24 well Cryschem Plate</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-158</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dow Corning Vacuum Grease</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-510</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Siliconized glass circle coverslides</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-231</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>100% paraffin oil</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-411</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>1.88 inch wide Crystal Clear Sealing Tape</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-511</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>96 Well Imp@ct Plate (Microbatch plate)</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-098</td>
<td>&nbsp;</td>	</tr>	</tbody>
		</table>

protein crystallization for x-ray crystallography

使用生物大分子的三维结构来推断它们如何运作，是现代生物学的最重要领域之一。原子分辨率结构的可用性提供了一个深刻和独特的理解蛋白质的功能，并有助于揭示活细胞的内部工作。迄今为止，86％的蛋白质数据银行（RCSB PDB）项的确定，采用X射线晶体学的大分子结构。 为了获得适合晶体，晶体学研究，大分子（如蛋白质，核酸，蛋白质 - 蛋白质复合物或蛋白质 - 核酸复合物）必须得到净化同质化，或尽可能接近同质化。同质化的准备是获得晶体衍射高分辨率（Bergfors，1999年;麦弗逊，1999年）的一个关键因素。 到过饱和的高分子结晶需要。样本，因此，应集中到尽可能高的浓度，而不会造成聚集或沉淀的高分子（通常为2-50毫克/毫升）。样品引入沉淀剂，能促进蛋白质晶体的成核的解决方案，这可能会导致大从溶液中的三维晶体生长。获得晶体主要有两种技术：气相扩散和间歇结晶。一滴含有沉淀和蛋白质溶液的混合物蒸气扩散，是密封在一个纯粹的沉淀室。水蒸汽，然后扩散的下降，​​直到渗透压下降，沉淀的是平等的（图1A）。脱水下降导致蛋白质和沉淀缓慢的浓度，直到实现平衡，坐落在相图的晶体核区。批处理的方法依赖于使混合蛋白的蛋白质直接进入核区与适量的沉淀（图1B）。这种方法通常是下一个石蜡/矿物油混合，以防止下拉水的扩散。 在这里，我们将演示两种蒸汽扩散实验装置，挂在下降，坐滴，除了批量下油的结晶。

Crystallization is usually referred to as the bottleneck of X-ray crystallography. A sparse matrix incomplete factorial screen of precipitating conditions typically produces many different types of protein aggregation and precipitation, among them large single crystals. If the protein or precipitant concentrations are too high one can see brown matter with no distinct shape and size (amorphous precipitation). When the solution is undersaturated, the drop will often be completely clear and devoid of any kind of precipitat...

Watch this Scientific Journal Video about Protein Crystallization for X-ray Crystallography at JoVE.com

Protein Crystallization for X-ray Crystallography

一个分子的三维结构的分子的功能提供了一个独特的理解。在近原子分辨率的结构测定的主要方法是X射线晶体学。在这里，我们展示了取得任何给定的高分子三维晶体，通过X射线晶体结构的决心适合的电流的方法。

X射线晶体学的蛋白质结晶

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

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63.0K 次观看.  Yale University. 一个分子的三维结构的分子的功能提供了一个独特的理解。在近原子分辨率的结构测定的主要方法是X射线晶体学。在这里，我们展示了取得任何给定的高分子三维晶体，通过X射线晶体结构的决心适合的电流的方法。

视频: Protein Crystallization for X-ray Crystallography

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

非侵入性的三维可视化与亚微米分辨率使用同步加速器X射线断层扫描

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

评估电子晶体结构生物学研究的小膜蛋白的二维结晶试验

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

基础X射线计算机断层数据的三维打印设置

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

提高蛋白质结晶的成功率随机Microseed矩阵筛选

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...