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。

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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 ...

automated-hydrophobic-interaction-chromatography-column-selection-for

Nanyang Technological University

Research

JoVE Journal

Biology

43.4K 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

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 ...

科研

生物学

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 ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）:一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104纯化，蛋白质Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...