Name: separation of bioactive small molecules, peptides from natural sources and proteins from microbes by preparative isoelectric focusing (ief) method
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Description: Watch this Scientific Journal Video about Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing IEF Method at JoVE.com

我们感谢生物学司和强生癌症研究中心分别为GV颁发BRIEF和IRA奖的资金来源。我们还感谢K-INBRE博士后奖RV。这项工作得到了国家卫生研究院国家普通医学研究所的机构发展奖（IDeA）的部分支持，该奖授予P20 GM103418。内容完全由作者负责，不一定代表国家普通医学研究所或国家卫生研究院的官方观点。我们感谢匿名评论者提供的有用意见。

1. 标准液相 IEF 单元的设置和预运行
<ol><li>根据说明手册（参见材料表），将液相 IEF 电极（阳极红色按钮和阴极黑色按钮）与各自的交换膜组装。使用 0.1 M NaOH 对离子交换膜进行平衡，在使用新膜时，阳离子交换膜与 0.1 M H3PO4交换膜至少 16 小时。<ol>
<li>在运行之间将膜存放在电解质中（0.1 M NaOH 或 0.1 M H3PO4），不允许干燥。</li>
	</ol>
</li>
	<li>通过在...

<ol>
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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+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72, 248-254 (1976).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Riazi, S., Dover, S., Turovskiy, Y., Chikindas, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commercial+ampholytes+used+for+isoelectric+focusing+may+interfere+with+bioactivity+based+purification+of+antimicrobial+peptides.">Commercial ampholytes used for isoelectric focusing may interfere with bioactivity based purification of antimicrobial peptides.</a> Journal of Microbiological Methods. 71 (1), 87-89 (2007).</li><li>Kamei, K., Takano, R., Miyasaka, A., Imoto, T., Hara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino-Acid-Sequence+of+Sweet-Taste-Suppressing+Peptide+(Gurmarin)+from+the+Leaves+of+Gymnema-Sylvestre.">Amino-Acid-Sequence of Sweet-Taste-Suppressing Peptide (Gurmarin) from the Leaves of Gymnema-Sylvestre.</a> Journal of Biochemistry. 111 (1), 109-112 (1992).</li><li>Craik, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry.+Seamless+proteins+tie+up+their+loose+ends.">Chemistry. Seamless proteins tie up their loose ends.</a> Science. 311 (5767), 1563-1564 (2006).</li><li>Craik, D. J., Daly, N. 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来自天然产品来源（如植物）的小分子包括复杂的二次代谢物，在化学结构上差异很大。据信，他们参与了植物防御机制。此外，多肽也存在于植物组织22。这些天然产物小分子是药物发现和开发测试分子的丰富来源。然而，其隔离和纯化所需的困难和繁琐的方法限制了它们用于治疗应用。本报告中使用的液相IEF方法强调了在不损害其生物活性的情况下分离这些小分子和多肽的?...

从复杂的生物样品中纯化生物分子是生物实验中必不可少的一步，而且往往是困难的步骤。同电对焦（IEF）非常适合复杂生物分子的高分辨率分离，其中载体放大物根据其电荷移动，并在电场3中建立pH梯度。IEF的第一个商业载体安培利特于1964年由奥洛夫·韦斯特伯格开发，并申请了44，5的专利。载体安皮质是脂肪寡聚-氨基-碳水化合物酸分子，长度不同，分支6。随后，韦斯特伯格等人改进了载体增强剂，以扩大在分离生物分子66、77中的用途。
分离生物分子的方法包括阿甘丝和多丙烯酰胺凝胶电泳、二维凝胶电泳（2-DE）、异电聚焦、毛细血管电泳、异丙三酚酶和其他色谱技术（如TLC、FPLC、HPLC）2。2液体相IEF在一种叫做"罗托福"的仪器中执行，是由米兰比尔8发明的。他开创了该仪器的概念和设计，为电泳迁移理论做出了广泛贡献。他的研究小组还开发了一个用于计算机模拟的电泳分离过程数学模型。
液相IEF装置是一种水平旋转圆柱形电池，由尼龙芯组成，分为20个多孔隔间和一个循环水冷却陶瓷棒。多孔腔允许分子在电极之间的水相中迁移，并允许在真空下以分数收集纯化样品。此净化系统可在 <4 小时内提供高达 1000 倍的特定分子纯化。该仪器的一个有价值的特点是，它可以作为从复杂混合物中纯化的第一步，或作为实现纯度10的最后一步。如果感兴趣的分子是蛋白质，另一个好处是其原生构象将在分离过程中保持。
液体相IEF的使用被广泛报道用于蛋白质、酶和抗体纯化66、10、11、12、13、14。,10,11,12,13,14在这里，我们描述了这种方法用于分离和纯化小分子和肽从药用植物Gymnema sylvestre。该协议将帮助研究人员集中和纯化活性小分子从植物提取物下游应用低成本。此外，我们还演示了从这个基于IEF的系统中从念珠菌藻真菌中提取的复杂蛋白质中提取的蛋白质富集15作为第二个例子。

<table>
	<tbody>
		<tr>
			<td>0.45 &#181;m syringe filter</td>
			<td>Fisher scientfic</td>
			<td>09-720-004</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>207861-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Lyte 3/10 Ampholyte</td>
			<td>Bio-Rad</td>
			<td>163-1113</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Lyte 5/8 Ampholyte</td>
			<td>Bio-Rad</td>
			<td>163-1192</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact low temperature thermostat</td>
			<td>Lauda -Brinkmann</td>
			<td>RM 6T</td>
			<td>Set water cooling to 4 oC and it can be run even at 0 oC as when it passes through the Rotofor cooling core, the circulating water temperature will be around 5 or more depending on the voltage.</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R</td>
			<td>Sigma-Aldrich</td>
			<td>B7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing (3,500 MWCO)</td>
			<td>Spectrum Spectra/Por</td>
			<td>132112T</td>
			<td></td>
		</tr>
		<tr>
			<td>Gymnema plant leaf extract powder (&#62;25% Gymnemic acids)</td>
			<td>Suan Farma, NJ USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Lab companion</td>
			<td>SI 300R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM 6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini protean electrophoresis</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Mettler Toledo</td>
			<td>S20</td>
			<td>Useful to determine the pH of the Rotofor (liquid-phase IEF) fractions</td>
		</tr>
		<tr>
			<td>Rotofor</td>
			<td>Bio-Rad</td>
			<td>170-2972</td>
			<td>http://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1702950E.pdf (Rotofor Instruction manual for assembling the unit)</td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>HyClone</td>
			<td>DH30255.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealing tape</td>
			<td>Bio-Rad</td>
			<td>170-2960</td>
			<td>Scotch tape may also be used.</td>
		</tr>
		<tr>
			<td>Sorvall legend micro 17 centrifuge</td>
			<td>Thermo scientific</td>
			<td>75002432</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP tissue culture plate -96 well flat bottom</td>
			<td>TPP</td>
			<td>TP92696</td>
			<td></td>
		</tr>
	</tbody>
</table>

separation of bioactive small molecules, peptides from natural sources and proteins from microbes by preparative isoelectric focusing (ief) method

植物和微生物的天然产物是生物活性分子的丰富来源。在使用之前，复杂提取物中的活性分子必须纯化，以便下游应用。有多种色谱方法可用于此目的，但并非所有实验室都能提供高性能方法，与复杂的生物样品隔离可能很困难。在这里，我们演示了预置液相异电对焦（IEF）可以根据复杂的植物提取物（pI）分离分子，包括小分子和肽。我们采用了该方法进行复杂的生物样品分馏和表征。作为概念的证明，我们分馏了一种Gymnema sylvestre植物提取物，分离出一系列二苯丙酮皂素小分子和肽。我们还演示了使用念珠菌藻菌作为模型系统的有效微生物蛋白分离。

从Gymnema sylvestre植物提取物分离和纯化小分子和肽 采用预选液相IEF方法，从人类致病性真菌C.白化病菌中分馏药用植物提取物和细胞表面蛋白。图 1显示了这些分馏协议的原理图。
从液相IEF获得的G.sylvestre提取物的20个分数中，发现暗色分子（二烯类皂苷）在阳极端（pH 2-3）迁移和浓缩，在阴极端（pH 8-9）观察到浅黄色?...

Watch this Scientific Journal Video about Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing IEF Method at JoVE.com

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing IEF Method

目标是通过使用液相同电聚焦（IEF）方法，分馏和分离生物活性小分子、复杂植物提取物中的肽和致病微生物中的蛋白质。此外，分离的分子被识别，其生物活性得到确认。

通过预置等电聚焦（IEF）方法，将生物活性小分子、肽与天然来源和蛋白质从微生物中分离出来

Natural products derived from plants and microbes are a rich source of bioactive molecules. Prior to their use, the active molecules from complex extracts ...

我们的协议描述了植物提取物中活性分子分离和纯化的新发现。科学家可以使用这些方法将小分子和肽从天然产品来源分离给治疗使用者。熟悉仪器部件及其装配方法。请务必确保要分离的样本由建议的准则编写。此方法涉及仪器的多个装配过程。视觉演示将帮助用户跟踪并成功地将其应用于研究。组装液相后，IEF单元如说明书所述，将阳离子交换膜与0.1摩尔氢氧化钠和阳离子交换膜与0.1摩尔磷酸平衡。对齐橡胶垫片，使三个大长方形孔保持橡胶疏通。这将有助于在电泳期间通过膜在电极和样品室之间交换离子。通过对齐离子交换垫片中的三个长方形孔，组装电极的内侧和外侧。然后用各自的电解质填充电极，以防止其膜干燥。接下来，拧紧塑料螺钉支架，以确保无泄漏组件。用密封胶带盖住样品收集板。接下来，将聚焦室的部件按以下顺序组装在陶瓷冷却手指上：阳极电极、尼龙膜芯、聚焦室和阴极电极。使用 50 毫升注射器，将聚焦室充满预冷却蒸馏水。对于标准 IEF 电池，加入 60 毫升水。将 IEF 装置连接到四摄氏度的循环冷却水。以 15 瓦和 3000 伏运行装置，直到电压稳定，大约 3 到 5 分钟。然后，关闭电源。使用分数收集器，从单元格中去除水。用密封胶带重新密封收集板。加入0.6克金尼玛西尔维斯特植物提取物，到60毫升蒸馏水。在辊管中混合五分钟，溶解植物提取物。要去除不溶性颗粒，请将溶液在10，000倍G下离心5分钟。将上半液转移到 150 毫升玻璃烧杯中，并加入安磷酸盐，按体积（约 0.6 毫升）创建 1% 溶液。使用 50 毫升注射器和 1.5 英寸 19 量表钝端针，通过样品采集板将该溶液加载到 IEF 电池中。然后从支架上取下电池，然后轻点电极室，以清除并清除任何气泡。将设备连接到冷却水。随着电源恒定的 15 瓦开始分馏。运行 IEF 单元，直到电压达到恒定值（大约三个小时）。按下"收获"按钮后，将分数收集器引脚与收集板对齐。将针脚穿过密封胶带，开始收集金尼玛西尔维斯特分数。在30摄氏度下在30摄氏度下生长一个C.albicans和酵母辣椒汤的单个殖民地。在震动下。将培养素转移到离心管中，以10000次G离心收集酵母细胞，5分钟。取出上经剂后，将酵母细胞悬浮在缓冲液中，并在滚管中旋转悬浮液一小时，温度为5摄氏度。要去除酵母细胞，将悬浮液在10，000次G下离心5分钟。然后通过0.45微米过滤器过滤蛋白质提取物。将蛋白质提取物转移到透析管中，在4摄氏度下对水进行透析15小时。在估计蛋白质浓度后，收集含有500毫克总蛋白质的蛋白质提取物。在60毫升水中稀释500毫克蛋白质，体积含1%的磷酸。使用注射器将稀释的蛋白质提取物加载到 IEF 细胞中，并在恒定的 15 瓦下操作装置 4 小时。由于此仪器使用高压电源，因此用户必须采取一切预防措施，避免与带电电极直接接触。分数收集器包含 20 个塑料管，每根 12 毫米由 75 毫米。它连接到真空泵。通过按下按钮上的收获准备收集分数。然后将分数收集器上的 20 个收集引脚与 20 个收集板对齐，以便密封对焦单元。将收集销通过密封胶带，同时打开真空泵。每个管子将收集大约三毫升的蛋白质分数。在减少和煮沸蛋白质分数后，在12.5%SDS页面凝胶上分析它们。用库马西蓝色染料染色凝胶，并在室温下放在摇杆上。两到三个小时后，将凝胶淡化。然后使用凝胶成像器记录凝胶图像。在RPMI细胞培养基中以1：1000的比例稀释C.albicans的隔夜培养，并辅以50毫摩尔葡萄糖，准备酵母细胞悬浮液。将 90 微升的电池悬浮液添加到 96 井板的每个井中。接下来，将每个 G.sylvestre 分数的 10 微升添加到单独的井中。作为负对，将10微升水与1%的磷酸盐加到单独的井中。在37摄氏度下孵育96井板12小时后，在显微镜下观察该板。缺乏纤维生长表明C.albicans酵母到催眠转化的抑制。通过液相 IEF 获得 20 部分 G.sylvestre 提取物。深色分子，健身房酸，迁移到和丰富在单位的阳极端。在阴极端观察到半透明浅黄色分数。每个 G.sylvestre 折射的等量通过 SDS 页面减少、煮沸和解析。库马西蓝色污渍显示不同的多肽带在约5千陀吨，丰富在分数16至19。第一分数中的金酸和接下来的几分之一没有检测到，因为它们不是蛋白质。然而，这些分子可以通过TLC分离，并在紫外光下检测。分数10不包含这些小分子的可检测量，这表明大多数有机小分子在一到三分数中被丰富。所有20G.sylvestre分数被检测为抑制酵母到催眠转化和催眠生长在C.albicans。在分数 1 中观察到最大的抑制，在分数 10 及以上观察到很少或没有抑制。使用液相 IEF 对来自 C.albicans 的细胞表面蛋白进行分馏。SDS 页面分析了 18 个分数的等分。丰富的蛋白质在几个分数中可见。开始时，小分子不能通过等电聚焦来分离，因为它们被认为是非流动的。我们的研究表明，它们是流动的，至少每周一次，而且这种方法可以进一步探索其他小分子。

Research

JoVE Journal

Immunology and Infection

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

科研

免疫与感染

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

定量成像小样本人体血液中单核细胞和树突状细胞系特定的Toll样受体介导的信号

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

分离<em&gt;恶性疟原虫</em&gt;晚期感染的红细胞由磁性装置

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

自动分离<em&gt; C。线虫</em&gt;可多段由细菌病原体殖民者

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

性T细胞Transinfection从树突状细胞捕捉细菌

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...