Name: total protein extraction and 2-d gel electrophoresis methods for burkholderia species
Uploaded: 2013-10-15T17:30:00
Description: Watch this Scientific Journal Video about Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species at JoVE.com

这项工作是由英属哥伦比亚大学（至BV）的一个助学金，并从健康研究囊性纤维化加拿大和加拿大学院（存保计划）资助。我们感谢韦涌协议的前期准备工作。

1。文化增长（天1 +2） <ol><li>成长起始培养：1毫升卢里亚BERTANI（LB）肉汤在一个单元前15 ml管，在37℃旋转过夜。稀释过夜生长至OD 600为0.6。加1ml该稀释液在250毫升的锥形烧瓶中百毫升的LB肉汤中。在37℃，250rpm振荡16小时为固定相（SP），以更好地近似，在分批培养，感染的条件，并确保细菌的毒力因子固定相的信号因子如RPOS和法定人数的控制下传感，表示。可替代地，在早期的SP或中?...

<ol>
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Biol. 424, 35-42 (2008).</li><li>von der Haar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protein+extraction+for+quantitative+proteomics+of+yeasts.">Optimized protein extraction for quantitative proteomics of yeasts.</a> PLoS One. 2, e1078 (2007).</li><li>O'Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+two-dimensional+electrophoresis+of+proteins.">High resolution two-dimensional electrophoresis of proteins.</a> J. Biol. Chem. 250, 4007-4021 (1975).</li><li>Gorg, A., Weiss, W., Dunn, M. 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一种蛋白质的制备方法进行了描述，可以提取大多数伯克霍尔德杆菌蛋白具有良好的再现性。这表现在从使用相同的细菌培养在LB或YEM肉汤生长， 如图1中不同的日子进行的两个独立的制剂获得相同的蛋白质谱。提取是有效的在液体培养基中生长的细菌，但是我们没有测试这个方法对于生长在平板上的细菌。也用这种方法作进一步鉴定蛋白质利用蛋白质组学工具;使用2-D凝胶电泳...

伯克氏菌属包括超过62种，从众多的壁龛中分离革兰氏阴性有机体，它是分为两个主要集群1,2。第一组包括人类，动物和phytotrophic生物，大多数研究都集中在这个群体的致病物种，由于其重要的临床意义。最致病的成员是B。杆菌和B。单孢菌 （它们分别引起鼻疽和鼻疽）3,4和机会致病菌5（对洋葱伯克霍尔德杆菌复合物，基底细胞癌的17中定义的物种），这引起疾病囊性纤维化（CF）和慢性肉芽肿病（CGD）1。第二组，有超过30种致病性，包括与植物或与环境有关的细菌，并且被认为是主机2具有潜在的好处。 许多并发症出现FR嗡细菌感染与伯克霍尔德杆菌属的致病成员，如因为抗生素使得难以在大多数情况下6-9根除固有或获得性耐药性的患者，该疾病的传播和治疗失败的病原体的传播。因此，获得的基础，建立细菌感染更清楚的了解，是造成这些疾病的生物治疗是至关重要的。为了深入了解建立感染，因此需要对与发病有关的细菌成分深入调查。研究集中于伯克霍尔德生物体的蛋白质组分析使用蛋白质组学方法描述了被认为与细菌致病以及改变其蛋白质型材10-16蛋白质。 用超声处理并冷冻解冻循环中裂解缓冲液含有高浓度的蛋白质提取方法尿素entration，硫脲在用洗涤剂和两性电解质结合，在鼻疽蛋白质组学研究10-13得到了应用。虽然尿素是蛋白质变性非常有效，它可以建立平衡水溶液中与异氰酸铵，它可以与氨基酸基团反应，从而形成伪影（氨甲酰化反应）17。因此，建议以包括载体两性电解质，它作为氰酸酯清除剂，并避免温度超过37℃17。此外，为了防止裂解缓冲液与蛋白质定量的任何化学干扰，相同的裂解缓冲液，可用于生成标准曲线，以使样品和标准品具有相同的背景10。其他方法包括使用碱性缓冲液和洗涤剂用热潜伏期17,18，但这些条件可能会导致改变蛋白质和某些清洁剂不与公关兼容oteomics申请，除非后续的清洁剂去除步骤包括17,18。 经过适当的提取和定量，每个单独的蛋白质的全球蛋白表达可以使用蛋白组学方法，如二维（2-D）凝胶电泳进行研究。这种技术首先由O&#39;Farell 19所述，由在蛋白质根据它们的等电点在第二维分离通过等电聚焦中的第一维度，然后根据它们的分子量通过丙烯酰胺凝胶电泳。由于其分辨率和灵敏度，该技术是一种强大的工具，用于从复杂的生物源19,20的蛋白质的分析和检测。这种分离技术是目前可用的蛋白质为中心的方法与解决引起转录后修饰或蛋白水解处理的蛋白异构体的巨大优势。量的变化可以通过凝胶20的染色后比较相应的点的强度进行检测。然而，这种技术不适合于非常大的蛋白，膜蛋白，极碱性和酸性的或疏水的蛋白质的鉴定，并且是比较费力和耗时的技术20。新的多肽-中心的方法（非凝胶型），它们更健壮和目标变得可用，并且可以被用于通过差动稳定同位素标记方法，例如通过同位素编码的亲和标记的半胱氨酸标记（ICAT）21，和氨基的定量比较标签通过同位素标记相对和绝对定量（的iTRAQ）22。使用一个单一的蛋白质组学技术可能给信息不足，因此使用两个互补的蛋白质组学方法是必要的蛋白质组最充分评估的变化。然而，二维凝胶电泳被广泛使用，并可以常规运用的全titative在不同的生物体的几种蛋白质的表达。 在这里，我们描述了一个全细胞蛋白提取及二维电泳程序进行了调整和优化，从GE Healthcare的2-D电泳原理和方法手册80-6429-60AC 2004（ 鼻疽种<a href="http://www.amershambiosciences.com" target="_blank">www.amershambiosciences.com</a> ）。蛋白质进行提取使用珠打浆机用玻璃珠在PBS中含有5mM EDTA（乙二胺四乙酸）和1mM PMSF（苯甲基磺酰氟）存在下进行。这个程序允许以最小的降解蛋白质的量化和可修正蛋白质组学方法如前呈报15,23,24。 2-D凝胶电泳进行了用24个厘米长的固相pH梯度4-7和蛋白质根据其等电点分离。那么蛋白质根据其molec分离ular重量通过SDS-聚丙烯酰胺凝胶。此外，我们描述了用于可视化斑蛋白和银染方法，该方法是用于质谱分析兼容的银染方法。连同这些程序可以允许来自伯克霍尔德杆菌物种重要的蛋白质，可能参与发病的鉴定。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>Phenylmethanesulfonyl fluoride (PMSF)</td>
 <td>Sigma</td>
 <td>P7626</td>
 <td>Toxic, corrosive</td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>Fisher</td>
 <td>A18-1</td>
 <td>Flammable</td>
 </tr>
 <tr>
 <td>PBS Buffer</td>
 <td>Bioscience</td>
 <td>R028</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylenediaminetetraacetic acid (EDTA)</td>
 <td>Fisher</td>
 <td>BP118-500 </td>
 <td>toxic</td>
 </tr>
 <tr>
 <td>Glass beads (0.1 mm)</td>
 <td>BioSpec Products</td>
 <td>11079101</td>
 <td></td>
 </tr>
 <tr>
 <td>Nalgene Oak Ridge Centrifuge tubes, polycarbonate (50 ml)</td>
 <td>VWR</td>
 <td>21009-342</td>
 <td></td>
 </tr>
 <tr>
 <td>MicroBCA protein extraction kit</td>
 <td>Pierce</td>
 <td>23235</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge Tubes 2.0 ml conical Screw cap Tubes with Cap and O-Ring</td>
 <td>Fisher</td>
 <td>02-681-375</td>
 <td></td>
 </tr>
 <tr>
 <td>2-D Clean-Up kit</td>
 <td>GE Healthcare</td>
 <td>80-6484-51</td>
 <td></td>
 </tr>
 <tr>
 <td>Urea</td>
 <td>Invitrogen</td>
 <td>15505-050</td>
 <td>Irritant</td>
 </tr>
 <tr>
 <td>CHAPS</td>
 <td>Amersham Biosciences</td>
 <td>17-1314-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Dithiothreitol (DTT)</td>
 <td>MPBiomedical, LCC</td>
 <td>856126</td>
 <td>Irritant</td>
 </tr>
 <tr>
 <td>Immobiline DryStrips pH 4-7 24 cm</td>
 <td>Amersham Biosciences</td>
 <td>176002-46</td>
 <td></td>
 </tr>
 <tr>
 <td>Duracryl</td>
 <td>Proteomic Solutions</td>
 <td>80-0148</td>
 <td>Very toxic, carcinogen</td>
 </tr>
 <tr>
 <td>Ammonium persulfate</td>
 <td>Fisher</td>
 <td>BP179-100</td>
 <td>Flammable, toxic, corrosive</td>
 </tr>
 <tr>
 <td>N,N,N',N'-tetramethylethylenediamine (TEMED)</td>
 <td>Invitrogen</td>
 <td>15524-010</td>
 <td>Flammable</td>
 </tr>
 <tr>
 <td>Sodium dodecyl sulfate (SDS)</td>
 <td>Fisher</td>
 <td>BP166-500</td>
 <td>Acute toxicity, flammable</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Invitrogen</td>
 <td>15510-027</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Fisher</td>
 <td>HC1100-1GL</td>
 <td>Flammable, toxic</td>
 </tr>
 <tr>
 <td>Acetic acid</td>
 <td>Fisher</td>
 <td>A491-212</td>
 <td>Flammable, corrosive</td>
 </tr>
 <tr>
 <td>Glutaraldehyde</td>
 <td>Fisher</td>
 <td>G151-1</td>
 <td>Very toxic, corrosive, dangerous for the environment</td>
 </tr>
 <tr>
 <td>Potassium tetrathionate</td>
 <td>Sigma</td>
 <td>P2926</td>
 <td>Irritant</td>
 </tr>
 <tr>
 <td>Sodium acetate</td>
 <td>EM Science</td>
 <td>7510</td>
 <td></td>
 </tr>
 <tr>
 <td>Silver nitrate</td>
 <td>Sigma</td>
 <td>209139</td>
 <td>Corrosive, dangerous for the environment</td>
 </tr>
 <tr>
 <td>Formaldehyde</td>
 <td>Sigma</td>
 <td>252549</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td>Sodium thiosulfate</td>
 <td>Sigma</td>
 <td>S7026</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>EMD</td>
 <td>9230</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>MPBiomedical, LCC</td>
 <td>808831</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>MPBiomedical, LCC</td>
 <td>800688</td>
 <td></td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>Fisher</td>
 <td>A412-4</td>
 <td>Flammable, toxic, health hazard</td>
 </tr>
 <tr>
 <td>Sodium carbonate anhydrous</td>
 <td>EMD</td>
 <td>SX0400-3</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td>Mineral oil</td>
 <td>ACROS</td>
 <td>415080010</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>JA-20 ultracentrifuge rotor</td>
 <td>Beckman Coulter</td>
 <td>334831</td>
 <td></td>
 </tr>
 <tr>
 <td>Mini Beadbeater</td>
 <td>Biospec Products</td>
 <td>3110BX</td>
 <td></td>
 </tr>
 <tr>
 <td>Ettan IPGphor II Isoelectric Focusing System and accessories</td>
 <td>GE Healthcare</td>
 <td>80-6505-03</td>
 <td><a href="http://www.amershambiosciences.com" target="_blank">www.amershambiosciences.com</a></td>
 </tr>
 <tr>
 <td>Ettan DALT Large Vertical electrophoresis system</td>
 <td>GE Healthcare</td>
 <td>80-6485-27</td>
 <td><a href="http://www.amershambiosciences.com" target="_blank">www.amershambiosciences.com</a></td>
 </tr>
 </tbody>
</table>

total protein extraction and 2-d gel electrophoresis methods for burkholderia species

细菌种的细胞内蛋白水平的调查是重要的，以了解疾病引起这些微生物的致病机制。在这里，我们描述了从洋葱伯克霍尔德物种蛋白提取用玻璃珠在磷酸盐缓冲盐水乙二胺四乙酸和苯甲基磺酰氟的存在基于机械裂解的过程。这种方法可以用于不同的伯克霍尔德杆菌物种，不同的生长条件，并且很可能适用于其他细菌的蛋白质组学研究中使用。在蛋白质提取之后，一个二维（2-D）凝胶电泳蛋白质组技术进行说明，研究在这些生物体的蛋白质组全局变化。该方法包括蛋白质根据它们的等电点分子量的基础上，分离通过等电聚焦中的第一维度，随后分离通过丙烯酰胺凝胶电泳中的第二维度。分离的蛋白可视化是通过银染。

的蛋白图谱从相同的细菌培养物中提取的两种不同的场合比较分析表明，类似的模式绑扎表示成功的蛋白提取。提取的分子量的蛋白质从10-150 kDa的范围。 图的全细胞蛋白提取自鼻疽multivorans 1所示代表考马斯亮蓝染色凝胶（密件抄送的成员）的LB或酵母/甘露醇（YEM）肉汤和种植临床分离株从固定相收获。 对于二维凝胶电泳分析，200微克来自...

Watch this Scientific Journal Video about Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species at JoVE.com

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

在洋葱伯克霍尔德属的成员是重要的临床意义病原体。我们描述了一种菌体总蛋白的提取，采用机械破碎，和2-D凝胶电泳随后蛋白质组学分析。

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Research

JoVE Journal

Immunology and Infection

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

一个1.5小时的程序，鉴定肠球菌直接从血培养的物种

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

科研

免疫与感染

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

禽流感监测与FTA卡：实地方法，生物安全，和运输问题解决了

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

用于识别传染性朊病毒通过后通过一个鸟类的消化系统程序

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

巨噬细胞通过二维电泳蛋白组学分析

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

环介导等温扩增（LAMP）法进行的物种特异性检测<em&gt;艾美耳球虫</em&gt;可感染鸡

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

替代体外测定病毒衣壳结构完整性的方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization

原生聚丙烯酰胺凝胶电泳免疫溴分析内源性IRF5二聚化

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like ...