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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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凝胶电泳和同位素标记相对和绝对定量（的iTRAQ）蛋白质组技术15,23,24我们的小组已经成功地研究蛋白质组的变化。对于后者，蛋白质被萃取到含0.5M EDTA的PBS中，以避免PMSF的用的iTRAQ实验15的任何干扰的缓冲器。 重要的是要对物方舟，在蛋白质的提取工艺和2-D凝胶电泳，所有的步骤都要进行4°C或使用冰桶，以减少蛋白质分解，以及使用技巧堵塞和磨损丁腈手套，并涵盖所有寒冷的条件下皮肤，以防止任何斑点角蛋白污染削减了后续的质谱鉴定。当进行蛋白质提取，但同样重要的是考虑该PMSF具有短的半衰期时在水溶液中，根据制造商，因此，0.1mM的储备溶液在丙酮中，应在使用前立即进行。或者，其它的丝氨酸酶抑制剂或蛋白酶抑制剂是更可溶且稳定的，并且毒性较低的都可以使用，尽管我们没有测试它们在我们的实验中。 在此过程中使用的裂解缓冲液中不含尿素，以提取两水溶液（细胞质）的蛋白质和较少可溶性PROT这对增溶重要一型，包括一些膜蛋白17。但是，对于第一维等电聚焦（该协议的步骤3）样品制备过程中，将样品重新悬浮于含有尿素补液缓冲区。其他蛋白质组技术也涉及类似的待遇26。与补液步骤以下之前，似乎除去干扰物质或低分子量的杂质是用于高分辨率关键，我们建议使用2-D清洁试剂盒各蛋白样品中作为结果得到改善。以前装载样品第一维等电聚焦分离，确保有吸墨纸条持有人的权利大小的条状准备，因为这是有问题的削减在无菌的时尚大小合适当场（本步骤3.4协议）。 运行的2-D SDS-聚丙烯酰胺凝胶电泳之前，有必要确保该凝胶百分比适合所关注的预期大小（12.5％百分比凝胶可以解决蛋白质在14-100 kDa的范围内）。后第二维凝胶电泳完成后，蛋白质的可视化可以通过考马斯蓝或银染色来实现，后者是染色，检出限更敏感是低至0.1纳克/蛋白质/点20。可视化与银染蛋白质点都适合用质谱进一步分析。然而，重要的是要提一提，银染溶液不应含有戊二醛，因为这剂与蛋白质交联，因此它不与进一步的质谱分析20兼容。可替换地，检测和定量差异表达的蛋白质，可以使用另一种凝胶为基础的方法，例如二维差分在凝胶电泳（2D-DIGE）连同自动软件。这种方法采用了独特的荧光标记（ 例如，循环3,5和2），用于标记的样品和一个通用内部标准公关IOR到第二维电泳克服，在一定程度上，变异的2-D电泳27和再现性的缺点。此外，凝胶可与SyproRuby，这是一种有机金属钌螯合物污点设计用于蛋白组学应用的第二维后进行染色。它可以检测到蛋白质点具有相似的灵敏度是银染，但比考马斯蓝更高的灵敏度。染色后的凝胶可以拍到与激光扫描仪或透射28。 总之，这部影片描述了一个全细胞细菌蛋白质的提取方法和二维凝胶电泳的方法，可以让全球变化研究中的伯克霍尔德物种的蛋白质组。

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><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</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&amp;rsquo;,N&amp;rsquo;-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 colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt; </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>&lt;a href=&quot;http://www.amershambiosciences.com&quot; target=&quot;_blank&quot;&gt;www.amershambiosciences.com&lt;/a&gt;</td></tr><tr><td>Ettan DALT Large Vertical electrophoresis system</td><td>GE Healthcare</td><td>80-6485-27</td><td>&lt;a href=&quot;http://www.amershambiosciences.com&quot; target=&quot;_blank&quot;&gt;www.amershambiosciences.com&lt;/a&gt;</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微克来自类鼻疽杆菌总蛋白是使用（ 图2）。由于这种病菌是由美国疾病控制和预防25确认为B型生物战剂，蛋白质，编写程序，进行了生物安全3级实验室围堵，因此与标准作业程序。蛋白重悬浮于补液并施加到一个24厘米长的固相pH梯度4-7和蛋白质根据它们的等电点（pI）分离。然后条带被放置在SDS-聚丙烯酰胺凝胶和蛋白质根据它们的分子量（MW）的分离。随后，凝胶进行银染的蛋白点的可视化。结果表明，超过500个蛋白质点，可以通过质谱法进行个体识别的丰度。 <img alt="图1" fo:content-width="4in" src="/files/ftp_upload/50730/50730fig1.jpg" /> 图1。 洋葱伯克霍尔德multivorans的总蛋白质谱的菌株。蛋白的SDS-PAGE分析在从D-2214和D-2094菌株在LB或酵母/甘露醇（YEM）肉汤在两个不同的场合的固定相萃取（1和​​2）。 4微克蛋白被装在一个12.5％的凝胶的每个车道和彩色无线TH考马斯亮蓝。泳道M，蛋白质标志物。 <img alt="图2" fo:content-width="4in" src="/files/ftp_upload/50730/50730fig2.jpg" /> 图2。 类鼻疽杆菌 1234B二维凝胶电泳分析分离。代表银染2-D胶表现蛋白质分离的等电点4〜7范围的地带。全细胞蛋白提取物（200微克）在固定相用于分离在15％SDS-PAGE凝胶。

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

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

29.0K 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

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

二维凝胶电泳结合质谱法分析人垂体腺瘤组织蛋白质组

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

科研

癌症研究

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

热酚水提取纯化及革兰氏阴性菌脂多糖的可视化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

免疫与感染

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

共识脑源性蛋白，人类和小鼠脑蛋白质组使用两个2D-DIGE和Mini双向电泳免疫印迹法研究提取协议

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

神经科学

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

蓝原生聚丙烯酰胺凝胶电泳（BN - PAGE）分析，从细胞裂解液多蛋白复合物

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

生物学

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

质谱法富集细菌脂蛋白及 n-端脂肽的制备结构测定

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

免电力，顺序的核酸和蛋白质的分离

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol&#160;reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.

This study describes a new method of isolating murine brown adipocytes for gene and protein expression analysis.

从小鼠肩胛间棕色脂肪组织中分离棕色脂肪细胞以进行基因和蛋白质表达分析

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
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Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially all proteins in a cell lysate. This video will show the principles of 2D gel electrophoresis, a general procedure, and some of its biomedical applications.
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2D gel electrophoresis begins with IEF as the first dimension. Every protein has a pH value, called the isoelectric point or pI, where the net charge is zero. When a protein is subjected to an electric field, it will move toward the electrode with opposite charge. Samples of interest are loaded onto immobilized pH gradient, or IPG, strips which have embedded ampholytes, molecules containing both acidic and basic groups. An electric field is then applied to the pH gradient strip, causing the proteins to migrate until they reach the pH value matching their pI, where they lose their net charge.
Prior to running the second-dimension, the embedded proteins are treated with SDS, denaturing them and providing a uniform negative charge. Once completed, the IPG strips are placed onto a polyacrylamide gel. An applied electric field draws the proteins toward the anode, with larger proteins moving more slowly through the gel.
Once the protein mixture has been separated according to pI and molecular weight, the proteome map is visualized using stains, and proteins of interest are identified.
Now that we have discussed the principles of 2D gel electrophoresis, let&#39;s go over a typical laboratory procedure.
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Before the experiment can be performed, the proteins must be solubilized into media. Solubilization of the sample is achieved by de-aggregating proteins with a combination of chaotropic agents for disrupting hydrogen-bonding interactions, nonionic detergents to prevent altering of the proteins&#39; charge, reducing agents to break disulfide bonds, and buffers. To remove interfering abundant proteins and other molecules, the material is sequentially extracted by centrifugation, and collection of the resulting pellet; followed by treatment with endonuclease, an enzyme used to consume any DNA that would interfere with the experiment.
Once the proteins have been solubilized, IPG strips are prepared by rinsing with a strip-cleaning solution and left upside-down to dry. Each strip is then assigned a strip holder number. Once ready, the cellular extract is loaded onto the strips in a slow, sliding motion from the negative to the positive end. For the purpose of rehydration, damp blotting paper is placed on top of the electrode and under the gel strips; the IPG strips are then lined onto the IEF instrument. A high electric current is applied, and the proteins begin to migrate.
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Following completion of the first dimension, the gel for SDS-PAGE is prepared in a casting apparatus. The IPG strips are treated by placing them face down in SDS-containing equilibration buffer. The electrophoresis unit is readied with the addition of electrophoresis buffer. The treated IPG strips are collected using tweezers, placed on top of the gel plates, and sealed with agarose-sealing solution. A voltage source then applies an electric field, which is held until the fastest-moving proteins are 1 cm from the bottom of the gel.
After completion of the electrophoresis, the proteins must be visualized. Traditionally this is performed by staining with Coomassie blue or silver nitrate. Proteins of interest may be transferred from the gel, and analyzed by Western blot analysis.
A second identification approach involves excision of the proteins from the gel, digesting them, than analyzing them by mass spectrometry.
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Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the uses for 2D gel electrophoresis.
One of the most common uses for this technique is the identification of molecules involved in disease initiation and progression. 2D gel electrophoresis, coupled with mass spectrometry, can detect the up- or down-regulation of specific proteins in diseased areas in comparison to healthy ones.
Additionally, 2D gel electrophoresis is useful in following the progress of patients&#39; response to a potential therapeutic drug. Specimens may be taken from patients at various timepoints following administration of the treatment. In this way, 2D gel electrophoresis coupled with Western blot or mass spectrometry analysis, can detect proteins associated with negative responses such as inflammation; or the absence of proteins in an alleviated state.
Another use for 2D gel electrophoresis is in the study of protein structure and function following posttranslational modification, or PTM, which are additions to proteins following their translation from mRNA. PTM&#39;s can regulate a variety of functions, including protein signaling, gene expression, or cause oxidative damage. 2D gel electrophoresis is sensitive to modifications such as methylation or acetylation, which can cause a shift in pI as well as the molecular weight.
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You&#39;ve just watched JoVE&#39;s video on 2D gel electrophoresis. This video described the principles of the technique, a typical experimental procedure, and several of its applications in the field of biomedicine.
Thanks for watching!
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二维凝胶电泳

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct ...

教育

JoVE Science Education

化学

生物化学

SDS-PAGE Based Extraction of Extracellular Vesicles Associated Proteins: A Procedure to Extract Proteins from EVs and Prepare Them for In-Gel Digestion

Source: Lemaire, Q. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/60118/characterization-immune-cell-derived-extracellular-vesicles-studying">Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment.</a> J. Vis. Exp. (2020)
In this video, we demonstrate the SDS-PAGE in-gel digestion method for protein extraction from extracellular vesicles or EVs. Once isolated, the obtained protein fragments can be used for proteomic analysis.

基于 SDS-PAGE 的细胞外囊泡相关蛋白提取:从 EV 中提取蛋白质并准备用于凝胶内消化的程序

Source: Lemaire, Q. et al. Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment. J. Vis. Exp. ...

总蛋白提取和二维凝胶电泳方法

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