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或中期或晚期对数生长期的细菌生长，可用于获得对细菌适应相位信息。 </li><li>经过16小时测量外径600，确保文化已经用相同的条件下，相对于以前构造的生长曲线达到SP。 </li></ol> 2。蛋白抽提（3天） <ol><li&gt;冰寒在冰箱里所有的媒体及解决方案预先过夜。所有步骤都要进行与冰桶管。预冷微量离心并用Beckman Coulter公司的高性能离心分离机（或等同物），以4℃，并在转子内部（转子= JA-20，离心机= J2-HS）。 </li><li>准备新鲜的0.1M苯甲基磺酰氟丙酮（PMSF，174.19克/摩尔）溶液溶解0.0174克PMSF（&#39;小心&#39;PMSF是有毒和腐蚀性，使用面罩和手套）1毫升​​丙酮（&#39;小心&#39;丙酮是有毒和易燃，使用面罩和手套）。 </li><li>补PBS / EDTA / PMSF溶液:0.1毫升的0.1M的PMSF，0.1毫升0.5M的EDTA和9.8毫升的PBS。检查16小时培养物的OD大约是它应该在的SP。 </li><li>需要35毫升细菌培养和转移到橡树岭离心管和离心的文化在4500×g的20分钟，在4°C。 </li><li>除去上清液到一个废物容器及添加35毫升冷却的PBS和悬浮颗粒。再次离心机在4500×g的20分钟，在4°C。 </li><li>在1ml冷的PBS去除上清，重悬。转移到无菌2毫升的Eppendorf离心1分钟就高，在微量离心机以14,000×g下在4℃下取下并丢弃上清液。 </li><li>加入1 ml的PBS / EDTA / PMSF溶液，悬浮颗粒，并转移到2毫升的螺丝帽筒（带O型圈）含约0.5毫升的玻璃珠（预消毒）。加重悬沉淀物，以含有玻璃珠和珠抨击他们在4℃下在冷室1分钟的管子。做到这一点三倍，把样品在冰上每个庆典之后。 </li><li>离心机在14,000 xg下在微量离心1分钟。去除上清，把它放在一个无菌5毫升聚苯乙烯管。 </li><li>为了提高产量加1毫升的PBS / EDTA / PMSF解决同一管内再次包含玻璃珠。珠bash中的管在4℃下1分钟。难道这2个把样品置于冰上AF之三是bash。离心机以14,000×g离心对离心机一分钟，除去上清液，并加入到上清液从步骤2.8。用1ml注射器和一个0.20微米的过滤器将上清液从聚苯乙烯管中，以一个新的无菌2毫升Eppendorf管中。 </li><li>在这一点上，样品应测定蛋白质的量，使用Pierce MicroBCA蛋白提取试剂盒和200微克等分应直到需要在被冷冻-80℃。 </li></ol> 3。样品制备（4天） <ol><li>准备25毫升补液溶液（8M尿素，2％CHAPS，2％IPG缓冲液，0.002％溴酚蓝），并存储于2.5ml等分试样在-20℃下加7毫克二硫苏糖醇（DTT，154.2克/ mol）的使用到的补液原液2.5ml的等分试样之前。 </li><li>使用2-D清理试剂盒，在450微升的补液原液在步骤3.1制得的最终再悬浮过程解冻的样品从步骤2.10。 </li></ol> 4。第一维等电聚焦（IEF）（4天） 在本节中的所有步骤都使用的Ettan IGPhor IEF系统。 <ol><li>成立了IPG第一维条:用带清洁液清洁带人，然后洗用Milli-Q水，离开倒置晾干。每个样品分配到胶条固定号。 </li><li> （ - ）从年底从第二个更广阔的领域补液步加载示例。用干净的镊子拔胶条出来的包装。取出的胶条保护层。行去除粗糙面朝下在一个缓慢的，滑动相处的方式湿带（ - ）端（+）结束。 </li><li>把电极的顶部和下胶条湿面纸用无菌水，以防止烧坏。冲洗镊子轮用纯化水和乙醇在两者之间。除去所有气泡，薄覆盖与矿物油，以防止干燥出局。线机上的胶条持有人。 </li><li>运行12小时，在20℃下rehyd配给，1小时，在200伏，1小时，在500伏，1小时，在1,000 V，0.5小时，在4000伏，12小时，在8000 V，在300 V和24小时保持可以取出在这个时候。 </li><li> IEF结束后，建议进行第二向SDS-聚丙烯酰胺凝胶电泳中，除IPG胶条被冷冻在-80℃，在保鲜膜涂布有矿物油以供将来分析。或者，为了避免点条纹由于DTT接触，它可能包括的IPG胶条在第二维之前，使用包含碘乙酰胺的缓冲液的第二平衡步骤。 </li></ol> 5。第二维SDS-聚丙烯酰胺凝胶电泳（第5天） 在本节中的所有步骤都使用的Ettan DALTsix电泳仪。 <ol><li>组装胶万向轮装置，彻底清洗所有部件，并确保凝胶施法者等级和灰色橡胶结合的润滑与硅凝胶。把黑擦ber的三角挡块在底部。然后，交替地将分离器和玻璃板具有较短板的玻璃面向前方。确保分离器的背面和正面。填充剩余空间用填料（约5张），所以顶部是平的。将前面板与在外面的红色尖端。使用夹收紧两边，然后拧紧螺丝底部。 </li><li>制备凝胶放入297.3 Duracryl毫升，（&#39;小心&#39;Duracryl是有毒的使用面罩和手套），147毫升的Tris缓冲液中的1.5M pH值8.8，和143.3毫升的Milli-Q水与含有真空尖的烧瓶中一搅拌棒。把盖在上面，并与所述真空20分钟，混合溶液在适中的速度。真空是用来脱气溶液中。使过硫酸铵溶液（APS 2,218.8克/摩尔）新鲜的10％的0.2克APS的2毫升Milli-Q水中。 </li><li>加入凝胶混合物的凝胶脚轮:当真空完成后加入6毫升10％十二烷基硫酸钠（SDS，228.38克/ mol）的向烧瓶的侧避免将更多的气泡在溶液中。添加黄芪多糖，搅拌均匀，加0.25 ml N，N，N&#39;，N&#39;-四甲基乙二胺（TEMED，116.20克/摩尔）（&#39;小心&#39;TEMED易燃使用面罩和手套），并搅拌均匀。 </li><li>通过在该装置的后面插入一个漏斗倒入凝胶混合物为凝胶脚轮。倾至短板下方一英寸。加入1.5毫升的水饱和的正丁醇中的凝胶的顶部，并包顶部与保鲜膜，以避免脱水。等待1小时，以确保完全聚合。 </li><li>检查凝胶:当凝胶做（检查瓶），通过一个接收器拆解凝胶施法者。检查凝胶的完整性，同时冲洗玻璃板用温水，不要让水进入板。倒出丁醇和水冲洗凝胶的顶部两次摆脱丁醇。再次装满水的上方，让如果凝胶不使用马上坐下。 </li><li>准备带:从准备平衡缓冲液冷冻机（6M尿素，75mM的Tris-盐酸pH值8.8，29.9％的SDS，0.002％溴酚蓝）。这可以在-20℃下是预先制备并储存在10毫升的等分试样将0.1g DTT的一个10毫升平衡缓冲液溶液。一个管可用于2 IPG胶条。将带材面朝下的凝胶上的缓冲液，并让坐30分钟。记住这到底是哪个。 </li><li>制备电泳单元（2-D分离装置）加入4.5升的1X电泳缓冲液（25mM Tris碱[121.1克/摩尔]，192 mM的甘氨酸[288.38克/摩尔]，和0.1％w / v的SDS [288.38克/摩尔]），以运行罐。这个缓冲区可用于多达3倍。转动电泳单元上。检查水冷机水位和水填满它，如果它是低。开启机器后，按模式来检查指定的温度，这应该是10°C。 </li><li>使1升2×电泳缓冲液和1升运行缓冲液为1x，并将其存储在4℃下使琼脂糖密封液:重量0.25克agaros的e和溶解在50毫升的1X电泳缓冲液。脉冲，每次15秒。 </li><li>设立电泳单元:使用镊子取出每个条带，并用干净纸巾去除多余的缓冲区两侧。请勿触摸条。倒出的水凝胶的顶部，并与1X运行缓冲线凝胶顶部。使用干净的镊子，然后将条上的长板的顶部，与凝胶一面朝向自己。添加1X缓冲条带润滑它。滑条进一步降低使用胶条。除去所有气泡在带钢和凝胶之间。 </li><li>加琼脂糖密封解决方案，以条带的顶部将其密封。重复该带的其余部分。将装有凝胶成凝胶支架玻璃板和下部进入凝胶罐中。确保在外部腔室中的1X缓冲水平是在把上室上之前指定的底线。 </li><li>将框架上缓冲液槽上的玻璃板的顶部，并确保它是所有的娃Ÿ向下按下底部向下。使用一个漏斗，并添加2倍运行缓冲液到上部腔室到中间线。使用一个漏斗，并添加1X运行缓冲液到外室，直到它到达最上面一行。将盖在上面，然后打开电泳单元和电源组上。 </li><li>运行过夜，52 V，96个毫安，5 W.检查当前是要通过寻找在银线靠近顶部的气泡。运行该凝胶直到领先行是1厘米的底部。 </li><li>蛋白质也可被使用的设备和从其他公司如BIORAD或霍弗试剂进行分析，根据制造商的说明进行操作。 </li></ol> 6。该凝胶银染的凝胶可视化（5-6天） <ol><li>准备一个修复溶液1（800毫升乙醇中，加入200ml乙酸，和1000毫升在通风柜的Milli-Q水倒入一个塑料桶）。解决好6凝胶。把所有的机器关闭，并采取了电泳单元和PU的整个中间部分LL它在下沉。也把白色的立在水槽。拉出含有2倍的运行缓冲液的顶部托盘。 </li><li>拆卸设备，并从玻璃板取出凝胶进入修复液1。凝胶可以通过切断角时，它们被从玻璃板上取下被识别，弯道的数量削减相应于在连铸机的凝胶顺序。 </li><li>放置在容器上的摇杆修复溶液和凝胶剂为至少1小时至过夜，在4℃下</li><li>转移的凝胶固定溶液2（20毫升50％的戊二醛溶液，600ml乙醇中，加入5g连四硫酸钾（302.46克/摩尔），136克乙酸钠（82.03克/摩尔）和Milli-Q水的2000毫升）和发生在一个摇杆1小时。 </li><li>洗凝胶4倍，每次15分钟的Milli-Q水。 </li><li> 30分钟或长达48小时染色的凝胶在硝酸银溶液（4克硝酸银（169.87克/摩尔），加入500μl甲醛，和Milli-Q水的2000毫升）中。 </li><li> 1啫喱分在Milli-Q水。 </li><li>传送到显影剂溶液（60克碳酸钠（105.99克/摩尔），300微升的甲醛，15毫克的硫代硫酸钠（158.11克/摩尔）和Milli-Q水至2,000毫升）5-30分钟，直到凝胶染色。 </li><li>将凝胶在终止液（100克Tris碱（121.14克/摩尔），40毫升乙酸中，并Milli-Q水至2000毫升）10分钟。 </li><li>进行存储，将凝胶转移到甘油溶液（40mL甘油或400毫升如果长期贮存需要，和1600毫升Milli-Q水）搅拌10分钟，然后用凝胶干燥器中干燥。 </li><li>凝胶可以使用成像系统如扫描仪/密度计，利用光透射或凝胶成像仪拍下染色后进行可视化。图像分析软件，如从BIORAD PDQuest 2-D分析可以被用于从样品中获得的蛋白的定量和定性信息。 </li></ol> 7。凝胶银染的质谱分析</P&gt; <ol><li>固定凝胶在50％甲醇，5％乙酸1小时。 </li><li>在50％的甲醇，至少10分钟，以洗涤过夜。 </li><li>在蒸馏水中3次10分钟清洗。 </li><li>敏化用0.02％的硫代硫酸钠（158.11克/ mol）的2×15分钟。 </li><li>洗涤3次中急冻蒸馏水3×10分钟。 </li><li>在4℃下至少20分钟至1小时以冷冻1％硝酸银溶液（169.87克/摩尔）浸没凝胶</li><li>洗涤2×1分钟，用蒸馏水冲洗。改变托盘/桶。 </li><li>发展0.04％的甲醛在2％无水碳酸钠（105.99克/摩尔），丢弃时，它会变成黄色比较快，有三个批次，并准备改变每5分钟。 </li><li>在5％乙酸洗涤，然后存储在1％乙酸。 </li><li>冲洗1.5ml离心管（持有拿到现货切口）与USP级乙醇和风干后才能使用。 </li><li>干净的镊子，刀片和表面用100％的甲醇。 </li><li>喷雾手套和表面用乙醇。 </li><li>消费点在流罩或洁净空气区域。点也可以切除使用自动点铣刀。 </li><li>把它们放在Eppendorf管，船舶在冰或保持冰箱。 </li></ol>

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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+of+surface+proteins+of+Burkholderia+pseudomallei.">The identification of surface proteins of Burkholderia pseudomallei.</a> Vaccine. 25, 2664-2672 (2007).</li><li>Madeira, A., Santos, P. M., Coutinho, C. P., Pinto-de-Oliveira, A., Sa-Correia, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomics+(2-D+DIGE)+reveals+molecular+strategies+employed+by+Burkholderia+cenocepacia+to+adapt+to+the+airways+of+cystic+fibrosis+patients+under+antimicrobial+therapy.">Quantitative proteomics (2-D DIGE) reveals molecular strategies employed by Burkholderia cenocepacia to adapt to the airways of cystic fibrosis patients under antimicrobial therapy.</a> Proteomics. 11, 1313-1328 (2011).</li><li>Zlosnik, J. E. A., Speert, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Mucoidy+in+Virulence+of+Bacteria+from+the+Burkholderia+cepacia+Complex:+A+Systematic+Proteomic+and+Transcriptomic+Analysis.">The Role of Mucoidy in Virulence of Bacteria from the Burkholderia cepacia Complex: A Systematic Proteomic and Transcriptomic Analysis.</a> J. Infect. Dis. 202, 770-781 (2010).</li><li>Riedel, K., Carranza, P., Gehrig, P., Potthast, F., Eberl, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+the+proteome+of+Burkholderia+cenocepacia+H111:+setting+up+a+2-DE+reference+map.">Towards the proteome of Burkholderia cenocepacia H111: setting up a 2-DE reference map.</a> Proteomics. 6, 207-216 (2006).</li><li>Weiss, W., Gorg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+solublization+buffers+for+two-dimensional+electrophoresis.">Sample solublization buffers for two-dimensional electrophoresis.</a> Methods Mol. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+two-dimensional+electrophoresis+technology+for+proteomics.">Current two-dimensional electrophoresis technology for proteomics.</a> Proteomics. 4, 3665-3685 (2004).</li><li>Gygi, S. P., Rist, B., Griffin, T. J., Eng, J., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+low-abundance+proteins+using+multidimensional+chromatography+and+isotope-coded+affinity+tags.">Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags.</a> J. Proteome Res. 1, 47-54 (2002).</li><li>Ross, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents.">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Mol. Cell Proteomics. 3, 1154-1169 (2004).</li><li>Velapati&#241;o, B., Limmathurotsakul, D., Peacock, S. J., Speert, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+differentially+expressed+proteins+from+Burkholderia+pseudomallei+isolated+during+primary+and+relapsing+melioidosis.">Identification of differentially expressed proteins from Burkholderia pseudomallei isolated during primary and relapsing melioidosis.</a> Microbes. Infect. 14, 335-340 (2012).</li><li>Chung, J. W., Speert, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+identification+and+characterization+of+bacterial+factors+associated+with+Burkholderia+cenocepacia+survival+in+a+murine+host.">Proteomic identification and characterization of bacterial factors associated with Burkholderia cenocepacia survival in a murine host.</a> Microbiology. 153, 206-214 (2007).</li><li>Rotz, L. D., Khan, A. S., Lillibridge, S. R., Ostroff, S. M., Hughes, 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=Public+health+assessment+of+potential+biological+terrorism+agents.">Public health assessment of potential biological terrorism agents.</a> Emerg. Infect. Dis. 8, 225-230 (2002).</li><li>Thon, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+proteomic+analysis+of+protein+changes+during+platelet+storage+requires+complementary+proteomic+approaches.">Comprehensive proteomic analysis of protein changes during platelet storage requires complementary proteomic approaches.</a> Transfusion. 48, 425-435 (2008).</li><li>Wu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+New+and+Emerging+Proteomic+Techniques.">In New and Emerging Proteomic Techniques.</a> Methods Mol. Biol. 328, 71-95 (2006).</li><li>Lopez, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+silver+stain+and+SYPRO+Ruby+Protein+Gel+Stain+with+respect+to+protein+detection+in+two-dimensional+gels+and+identification+by+peptide+mass+profiling.">A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling.</a> Electrophoresis. 21, 3673-3683 (2000).</li></ol>

作者宣称，他们有没有竞争的财务权益。

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 ...

total-protein-extraction-2-d-gel-electrophoresis-methods-for

Research

JoVE Journal

Immunology and Infection

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

28.9K 次观看.  University of British Columbia. 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。 

视频: 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

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

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

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

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

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

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.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

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 ...

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

两种异核体的形成方法，发现丙型肝炎病毒的制约因素

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

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

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

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

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

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

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

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

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

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

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

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

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

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 ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

从果蝇幼虫中提取包囊微生物感染及分析

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

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

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

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

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

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 ...

总蛋白提取和二维凝胶电泳方法

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