Name: automated hydrophobic interaction chromatography column selection for use in protein purification
Uploaded: 2011-09-21T13:00:00.000+00:00
Duration: 10 min 21 s
Description: Watch this Scientific Journal Video about Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification at JoVE.com

这项工作是由国家卫生部授予GM086822和国家科学基金会主要研究仪器授予在DBI-0960313研究院。笔者想感谢博士。乔恩宅唐娜·哈迪（Bio-Rad公司）和他们的技术专长，詹妮弗Loertscher（西雅图大学）。选择色谱试剂和补充仪器Bio-Rad公司提供的慷慨。

<ol>
	<li>Cummins, P. M., O'Connor, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography.">Hydrophobic interaction chromatography.</a> Methods Mol. Biol. 681, 431-437 (2011).</li><li>Nagrath, D., Xia, F., Cramer, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+modeling+of+nonlinear+hydrophobic+interaction+chromatographic+systems.">Characterization and modeling of nonlinear hydrophobic interaction chromatographic systems.</a> J. Chromatogr. A. 1218, 1219-1226 (2011).</li><li>To, B. C., Lenhoff, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography+of+proteins.+IV.+Protein+adsorption+capacity+and+transport+in+preparative+mode.">Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode.</a> J. Chromatogr. A. 1218, 427-440 (2011).</li><li>Huddleston, J. 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C., Banh, C., Brossay, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L.A+Protocol+for+the+Production+of+KLRG1+Tetramer.">L.A Protocol for the Production of KLRG1 Tetramer.</a> J. Vis. Exp. (35), e1701-e1701 (2010).</li><li>Murphy, P. J. M., Morishima, Y., Kovacs, J. J., Yao, T. P., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+dynamics+of+hsp90+action+on+the+glucocorticoid+receptor+by+acetylation/deacetylation+of+the+chaperone.">Regulation of the dynamics of hsp90 action on the glucocorticoid receptor by acetylation/deacetylation of the chaperone.</a> J. Biol. Chem. 280, 33792-33799 (2005).</li><li>Zeytuni, N., Zarivach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+the+M.+magneticum+strain+AMB-1+Magnetosome+Associated+Protein+MamA&#916;41.">Purification of the M. magneticum strain AMB-1 Magnetosome Associated Protein MamA&#916;41.</a> J. Vis. Exp. (37), 10-3791 (2010).</li><li>Kong, Y., Li, X., Bai, G., Ma, G., Su, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automatic+system+for+multidimensional+integrated+protein+chromatography.">An automatic system for multidimensional integrated protein chromatography.</a> J. Chromatogr. A. 1217, 6898-6904 (2010).</li><li>Camper, D. V., Viola, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+protein+purification.">Fully automated protein purification.</a> Anal. Biochem. 393, 176-181 (2009).</li><li>Hammon, J., Palanivelu, D. V., Chen, J., Patel, C., Minor, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+green+fluorescent+protein+screen+for+identification+of+well-expressed+membrane+proteins+from+a+cohort+of+extremophilic+organisms.">A green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms.</a> Protein Sci. 18, 121-133 (2009).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+green+and+red+fluorescent+proteins+to+teach+protein+expression,+purification,+and+crystallization.">Using green and red fluorescent proteins to teach protein expression, purification, and crystallization.</a> Biochem. Mol. Biol. Educ. 36, 43-54 (2008).</li><li>Queiroz, J. A., Tomaz, C. T., Cabral, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography+of+proteins.">Hydrophobic interaction chromatography of proteins.</a> J. Biotechnol. 87, 143-159 (2001).</li><li>McCue, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+and+use+of+hydrophobic+interaction+chromatography+in+protein+purification+applications.">Theory and use of hydrophobic interaction chromatography in protein purification applications.</a> Methods Enzymol. 463, 405-414 (2009).</li></ol>

选择色谱试剂和仪器的补充，Bio-Rad公司提供。

液相色谱技术已被证明为高度纯化的蛋白质，必要时进行免疫6，生化7，准备和结构8研究非常宝贵的。 HIC的净化方法往往需要经验的首选媒介的决心，并配体结构，配体密度，矩阵珠属性，所有已被证明影响色谱结果2，3。自动列侦察是一种有效的方法选择HIC的媒介，为后续的优化和蛋白质纯化4。自动列侦察方法可以很容易地适应各种HIC的蛋白质纯化策略。盐的浓度，选择盐，盐梯度和pH的变化可能进一步提高净化条件，改变这些基本参数的影响，此前已审查13,14。 HIC的洗脱液含有部分纯化的目标蛋白酶可以进一步纯化，利用互补的色谱技术，如离子交换层析（离子交换）或凝胶过滤/体积排阻色谱9,10。 由于其独特的光吸收和发射特性，GFP流出曲线可以使用行和运行后的方法确定。为此，该协议可以进一步进行调整重组绿色荧光蛋白融合蛋白的纯化，其中无关的靶蛋白与GFP的“标签”。 GFP标记的目标蛋白质，可以检测紫外线灯11和纯化采用各种色谱方法，包括如上所述的HIC纯化策略。 GFP的净化，也成为在现代蛋白质科学技术12教学生化实验室教学主食。 虽然使用大幅度减少稳健的色谱SY可以进行基本的HIC的蛋白质纯化干，这里介绍的仪器有一个明显的优势，以帮助取得了良好的效果。利用一个高度自动化的系统显着的好处包括：提高运行运行状况的可重复性，节省时间，并减少空气进入系统10推出的机会。系统控制的缓冲区的混合，这是由系统最大化的便利，允许增加缓冲区的准备和实验重复性的一致性。所有球探的动态样本循环吸引足够的样品负荷运行进一步确保被添加到每个列样品的均匀性，并允许连续运行不中断或手动重装。从样品到列循环控制进样降低变异可能发生的样品装载手册。双列选择阀允许连续样品运行，每次使用不同的抗HIC列，，无需，replumb系统。执行MUL<html> TI波长分析化验了一个独特的光度轮廓的一种蛋白质时特别有利。此外，以绿色荧光蛋白，细胞色素，黄素，和其他含血红素的蛋白质可能从这项技术中受益。电导率和pH监测设备允许实时实验条件的核查。分割分数洗脱收集允许改进的部分处理，使运行后的分析方法，需要一个最小的样品量（如酶联免疫吸附试验，SDS-PAGE电泳，免疫印迹和Experion微流体电泳）容易转移。第二部分收集脱机操作时需要手动同步，它允许最大的灵活性，馏分收集器的选择。 HIC的的球探列和蛋白质纯化利用这样一个强大的色谱系统最显着的缺点，包括初始时间和相关的预算支出与仪器购置和操作人员的培训。 T“这里提出的协议利用Bio-Rad公司DuoFlow层析系统。然而，也可以利用同样强劲，从其他制造商，如GE医疗ÄKTA前卫，仪表，能够产生相同的结果，甚至可以媲美色谱系统有独特的特点（如方法编程，组件的命名，运营商的偏好，和可扩展性的限制）前应考虑发起一个净化过程或仪器采集。

<table border="1"><tbody><tr><td>试剂名称</td><td>公司</td><td>目录编号</td><td>评论（可选） </td></tr><tr><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">生物DuoFlow探路者20系统</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">7602257</a> </td><td>包括系统的最大化，搅拌机，F10键工作站，AVR7-3阀，QuadTec紫外/可见光探测器流细胞，BioFrac的馏分收集，生物软件，入门套件</td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">AVR9-8流选择阀</a><html></td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7600408</a> </td><td>高压阀门，9口，8位，3500 psi（233杆）的限制，使用生物DuoFlow系统</td></tr><tr><td>分流阀SV3T 2 </td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td> 7600410 </td><td>电磁阀，3端口，2位，30磅（2条）的限制，使用生物DuoFlow系统</td></tr><tr><td>流分配器阀</td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td></td><td></td></tr><tr><td>tical LSR与parentCategoryGUID的= 22c495f1-36ff-44a3-9140-ee7e54d0b1ba“目标=”_blank“&gt; DynaLoop 25套件</td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0451&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7500451</a> </td><td>滑模活塞样品环套件，包括25的毫升DynaLoop滑动活塞循环，DynaLoop部分试剂盒（＃750-0450） </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">BioFrac馏分收集器</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">7410002</a> </td><td>两个分数山坳<html>讲师使用</td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">BioFrac微孔板下降头适配器</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">7410088</a> </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=741-0017" target="_blank">BioFrac酶标板适配器</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td>国家=美国＆LANG = EN的productID = 741-0017“目标=”_blank“&gt; 7410017 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">紫外线光模块</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">7500202</a> </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-1331&vertical=LSR&parentCategoryGUID=c4095dc1-9260-4431-b066-823b050048c8" target="_blank">卤素灯</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td> &lt;<html>一目标=“_blank”&gt; 7601331 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">Econo梯度泵</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">7319001</a> </td><td>梯度泵低压蛋白质纯化，包括油管和接头套件</td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224301" target="_blank">Experion系统</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td>bio-rad.com/prd/en/US/adirect/biorad？CMD = BRCatgProductDetail及产品ID = 224301“目标=”_blank“&gt; 7007001 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">的Experion Pro260分析套件</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">BIO-RAD</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">7007102</a> </td><td></td></tr><tr><td> HiTrap HIC的列选择套件</td><td> GE医疗集团</td><td> 28-4110-07 </td><td>包括7×1毫升HIC的媒体预包装侦察列</td></tr><tr><td> GE医疗的Bio-Rad公司色谱柱接头</td><td> GE医疗集团</td><td> 18111251和18111257 </td><td></td></tr></tbody></table>

automated hydrophobic interaction chromatography column selection for use in protein purification

对比其他色谱法净化的蛋白质（如凝胶过滤，亲和，离子交换），疏水作用层析（HIC）通常需要实验测定（以下简称为筛查或“侦察”），以选择最合适的色谱介质为净化一个给定的蛋白质1。这里介绍的方法，介绍了自动化的方法，一个一个被用于蛋白质纯化的最佳HIC的媒体侦察。 HIC的蛋白质和其他生物大分子的分离从对疏水性的差异为基础的原油裂解。类似的亲和层析（AC）和离子交换色谱法（离子交换），HIC的是能够集中感兴趣的蛋白质，因为它通过色谱过程的进展。 HIC的最佳适合净化的蛋白质，包括那些与疏水表面区域，并能承受接触到盐浓度超过2米弹药钌硫酸（（NH 4）2 SO 4计 ）。 HIC的往往选择作为一个缺乏亲和标签蛋白的纯化方法，从而为AC不合适，当IEx未能提供足够的净化。对蛋白质表面的疏水基暂时绑定到非极性配体耦合的惰性，不动的矩阵。蛋白质和配体之间的相互作用是高度依赖流经层析柱缓冲盐浓度，离子浓度高，加强蛋白质-配体相互作用，使蛋白动（即列内的约束）2。随着盐的浓度下降，消耗的蛋白质 - 配体相互作用，蛋白质再次成为移动和流出列。 HIC的几个媒体市售预包装列，每几个疏水性配体（如S-丁基，丁基，辛基，苯基）包含一个交叉链接到一个规范的琼脂糖珠在不同密度IFIC直径3。自动列侦察允许一个有效的方法确定HIC的媒体应为未来，更详尽的优化实验和蛋白质纯化运行4。 特定的蛋白质纯化这里是重组绿色荧光蛋白（GFP）;然而，这种方法可用于净化与一个或多个疏水表面区域的其他蛋白质适应。绿色荧光蛋白作为一个有用的模型蛋白，由于它的稳定性，独特的光吸收峰在397 nm，荧光，当暴露在紫外线光5。准备在高盐缓冲含有野生型绿色荧光蛋白的细菌裂解液，装入一个Bio-Rad公司DuoFlow中型高压液相色谱系统，吸附到的HiTrap含有不同HIC的媒体HIC的列。蛋白被洗脱列，并在线路和运行后的检测方法分析。缓冲混合，动态样本循环注射，连续山坳UMN选择，多波长分析，并分割部分洗脱液收集增加了系统的功能和实验方法的重复性。

Watch this Scientific Journal Video about Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification at JoVE.com

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

提出了一个自动化的方法，确定合适的疏水作用层析（HIC）在蛋白质纯化过程中使用的媒体。该方法利用一个中型高压液相色谱系统包括自动缓冲混合，动态样本循环注射，顺序列选择，多波长分析，并分割部分洗脱液收集。

自动疏水层析蛋白质纯化用柱选择

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

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43.5K 次观看.  Seattle University. 提出了一个自动化的方法，确定合适的疏水作用层析（HIC）在蛋白质纯化过程中使用的媒体。该方法利用一个中型高压液相色谱系统包括自动缓冲混合，动态样本循环注射，顺序列选择，多波长分析，并分割部分洗脱液收集。

视频: Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

科研

生物化学

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

使用多柱板适配器的经济性和多功能性高通量蛋白质净化系统

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

Chromatography-based Biomolecule Purification Methods

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase, which contains target-specific ligands.
This video serves as an introduction to size-exclusion and affinity chromatography, as well as the concepts that govern them. A step-by-step procedure for the purification of a histidine-tagged protein by immobilized metal affinity chromatography is described. Applications for both of these chromatographic methods in biochemistry and biomedical research are also profiled.
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&#34;Chromatography&#34; refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule&#39;s properties and activities can be determined. Each chromatographic technique has a different mechanism for separation, depending on the sample matrix and target compound. This video will focus on the principles and operation of two methods common to biochemistry: size-exclusion and affinity chromatography.
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Size-exclusion chromatography or SEC is based on the size of the compounds in the sample. A mobile phase containing the sample is added to a column with a porous material: the stationary phase. The molecules in the sample fall into 1 of 3 categories.
Molecules too large to enter the pores travel the shortest distance through the column. Any species with a molecular weight above this &#34;exclusion limit&#34; will exit the column at the same time. Molecules small enough to freely enter the pores will be retained the longest, and will exit the column together. The molecular weight allowing complete pore entry is the &#34;permeation limit&#34;.
Only molecules between these limits will be separated from one another, as they spend varying amounts of time diffusing into and out of the pores. Smaller molecules are retained longer on the column because they spend more time in the stationary phase, whereas larger molecules within these limits exit earlier.
Molecular weights across 1 to 2 orders of magnitude fall within these limits. Columns are chosen with this in mind, or multiple columns can be used in series if there is a wide range of desired compounds.
Now that you&#39;ve seen the theory of SEC, let&#39;s look at how it is carried out.
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To begin the SEC procedure, the column must be equilibrated with deionized water and chromatography buffer. Once prepared, buffer containing the sample is injected onto the column. The buffer is then pushed through at a low flow rate. A detector monitors what exits the column to determine the presence of the desired analyte. Large molecules with molecular weights above the exclusion limit exit the column at the same time. Small fractions from the column are collected in tubes. Each fraction is tested for the quality of the target molecule by gel electrophoresis or other analytical techniques.
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Now let&#39;s have a look at affinity chromatography or AC, one of the most efficient ways to purify proteins. Many biomolecules bind selectively to certain ligands-a property which AC utilizes by adhering a target-specific ligand to the stationary phase.
When the mixture flows through the column, the target molecules attach to the ligand, and the rest flow through. After the mixture has passed through the column, the target molecule can be collected through one of two elution methods based on specificity.
Biospecific elution can be said to a have a &#34;normal-&#34; or &#34;reverse-role&#34;. In normal-role biospecific elution, an agent is added that competes with the adhered ligand to bind with the target biomolecule.
In reverse-role biospecific elution, an agent competes with the target to bind to the adhered ligand. The second elution type, nonspecific elution, lowers the target-to-ligand binding by changing the solution&#39;s pH, ionic strength, or polarity. If a protein does not bind to a ligand that can be immobilized, the protein can be expressed containing a &#34;tag&#34;: short peptide sequences engineered to bind to the ligand.
One variety is immobilized metal ion affinity chromatography, &#34;IMAC&#34; for short, where an adhered metal ligand, like nickel or cobalt, binds to histidine residues on the modified protein. Through molecular biology techniques, target proteins are generated with repeating histidine residues, called a polyhistidine-tag, which binds to the metal via the imidazole side chain on histidine. Once bound, the protein can be collected with free imidazole via reverse-role specific elution and later used in a wide variety of downstream applications.&#160;Now that you&#39;ve seen the theory of affinity chromatography, let&#39;s look at an IMAC procedure in the laboratory.

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In IMAC, the stationary phase can be added directly to the mixture of mobile phase and sample, allowing the binding of the his-tagged protein. This slurry is then poured into the column, where the non-bound compounds drip into waste, while the slurry remains. The slurry container is rinsed to collect residual resin and sample, which is added to the column.
The resin is stirred to ensure the unbound components are free-flowing. Added wash buffer helps flush them away. Once all of the unbound components have been removed, the waste is replaced with a container to collect the target protein.
Buffer containing imidazole is added, stirred, and allowed to rest to unbind the target molecule. The imidazole binds to the metal, replacing and releasing the tagged protein. The freed protein is collected, and the imidazole step is repeated to ensure total collection. To further purify the sample, SEC can be run on the sample prior to analysis.
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Now that we&#39;ve seen the theory and procedure of these two techniques, let&#39;s look at some of the ways they&#39;re applied in the biochemical field.
A common reason to purify proteins is to study their role in disease. Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator protein, or CFTR. After growing the protein with a his-tag in yeast, both affinity and size-exclusion chromatography allow the isolation of the protein, followed by the study of its function.
In some instances, the presence of a polyhistidine tag can change a protein&#39;s structure, thereby affecting its function. Another common tag is maltose-binding protein or MBP, which will bind to bound amylose in a column. Maltose is then used to release the complex. The MBP can then be cleaved and removed with SEC to produce the pure desired protein.
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You&#39;ve just watched JoVE&#39;s video on size-exclusion and affinity chromatography. It covered the theory of the techniques, went over general procedures, and covered some of the uses of the techniques.
Thanks for watching!
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Size Exclusion and Affinity Chromatography

基于色谱的生物大分子分离纯化方法

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by ...

教育

JoVE Science Education

化学

Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate

Source: Pokhrel, A. et al. A <a href="https://www.jove.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018)
In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.

基于镍亲和层析的蛋白质纯化:一种从细菌细胞裂解物中纯化多组氨酸标签的重组蛋白的技术

Source: Pokhrel, A. et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018)
In this ...

JoVE Encyclopedia of Experiments

Biological Techniques

Chromatography Techniques

Butyl-Dependant Hydrophobic Interaction Liquid Chromatography: A Technique to Characterize Antibody-Drug Conjugates Based on Hydrophobic Interactions

Source: Oller-Salvia, B. <a href="https://www.jove.com/v/58066">Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction</a>. J. Vis. Exp. (2018).
This video demonstrates the hydrophobic interaction liquid chromatography-based characterization of antibody-drug conjugates. Using a butyl ligand-based chromatography column, the antibody-drug conjugates are characterized via hydrophobic interactions with the ligand.

丁基依赖性疏水相互作用液相色谱法:一种基于疏水相互作用表征抗体-药物偶联物的技术

Source: Oller-Salvia, B. Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal ...

An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Source: Elgundi, Z., et al. <a href="https://app.jove.com/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of antibody-containing fractions.

一种用于纯化单克隆抗体的亲和层析技术

1. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System
NOTE: The following procedure can ...

Immunology

Antibody-Based Technologies

Production and Purification

Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography

Source: Karch, C. P. et al., <a href="https://app.jove.com/v/60103">Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation.</a>&#160;J. Vis. Exp. (2019)
This video showcases the purification of histidine-tagged self-assembled protein nanoparticles or SAPNs using affinity liquid chromatography. The resulting purified SAPN fractions are suitable for vaccine development, holding promise for potential immunotherapeutic applications.

使用亲和层析纯化自组装蛋白质纳米颗粒

1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3)

	Mix 95 mL of ...

Immunotherapy

Immunogenics and Vaccine Development

Types Of Column Chromatography

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating parameters such as pH, temperature, or solvent affect the packing of the column material, thereby determining the purification efficiency. The choice of column material also plays an essential role in deciding the operating parameters and can be modified based on the proteins that need to be purified.
Gel Filtration Chromatography 
When the protein&#39;s chemical nature is unknown, gel filtration chromatography is used for purification based on size. Matrices such as agarose, polyacrylamide, dextran derivatives, or silica beads are used for low-pressure systems, whereas polymeric resins are used for higher flow rates. The varying degree of cross-linking of these polymeric materials determines the pore size of the matrix, which decides the size range of proteins that can be separated.
Ion Exchange Chromatography
Ion-exchange chromatography can purify proteins based on their net charge at a particular pH determined by their isoelectric point or pI. The net charge on matrix resin is used to purify oppositely charged proteins by replacing the cations or anions from the matrix resin with the charged proteins. The resins are categorized based on the type of ion that is exchanged &#8212; positively charged proteins replace cations from cation-exchange resins, and vice versa. Strong ion exchangers with stability over a wide range of buffer pH include quaternary ammonium, sulfonate, and sulfopropyl resins. In contrast, weak ion exchangers that are effective only in a narrow pH range include diethyl aminoethyl (DEAE) or carboxymethyl (CM) resins.
Affinity Chromatography
Affinity chromatography involves immobilizing protein-specific antibodies or enzyme-specific substrates to the column material, usually agarose beads. For example, a column with the immobilized antibody of choice is prepared to separate specific antigens. When the mixed antigen sample is passed through the&#160; column, the target antigens bind to the immobilized antibodies and separate from the mixture. To separate recombinant proteins tagged with enzymes such as beta-galactosidase, the corresponding substrate Isopropyl-&#946;-D-thiogalactopyranoside (IPTG) is immobilized on the column to purify the enzyme.
High-pressure liquid chromatography (HPLC) and gas chromatography (GC) are the most commonly used column chromatography techniques. In HPLC, the solvent flows at high pressure through a tightly packed column that improves efficiency. Gas chromatography analyzes volatile compounds by converting them to their gaseous forms upon heating. The volatilized sample is pushed through the column by inert gasses such as nitrogen and helium.

柱色谱的类型

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating ...

JoVE Core

细胞生物学

Unit 1

Analyzing Cells and Proteins

关键术语与概念

Anion-Exchange Chromatography-Based Protein Purification: A Separation Technique to Isolate a Protein of Interest From Dialyzed Bacterial Lysate Based on Net Charge

Start the FPLC with general maintenance. Connect column buffers AIEX-A and AIEX-B to buffer valves, and the anion-exchange resin-containing column to the column ports of the FPLC. Adjust the general chromatographic parameters accordingly.
Start the AIEX program in the FPLC software and choose the equilibration volume for buffer A. Subsequently, load the sample onto the column, and elute the proteins with a linear gradient from 0% to 100% high-salt buffer. Collect 2-milliliter fractions during elution.
Analyze the eluted fractions after finishing the run.

基于阴离子交换色谱的蛋白质纯化:一种基于净电荷从透析的细菌裂解物中分离目标蛋白质的分离技术

EoE Sub Articles

Calcium-Dependent Hydrophobic Interaction Chromatography: A Technique to Purify Calcium-Binding Proteins Based on Hydrophobic Interactions

To continue the protein purification, after the protein dialysis, add calcium chloride to the sample to a final concentration of 25 millimolar, which will facilitate binding of S100A12 to the resin in the next step. Then, filter the sample through a filter with a pour size of 0.45 microns.
Equilibrate HIC buffers and samples to 4 degrees Celsius. Next, connect column buffers HIC-A and B and the column, to the system and adjust the parameters.
Start the method to equilibrate the column with 1 to 2 column volumes of buffer HIC-A, and load the sample, and the &#39;wash unbound sample block&#39; when the UV signal reaches baseline level again.
Then, start elution with a calcium chelator-containing buffer EDTA. Collect 2 milliliters of peak fractions during elution.

自动疏水层析蛋白质纯化用柱选择

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