这项研究得到了美国国立卫生研究院的拨款:R21AG042066。我们要感谢菲利普·舒尔茨，他在创建屏幕捕获视频的贡献。

1.噬菌体生产<ol><li>执行中的生物安全柜的所有噬菌体的生产和生物淘洗过程。产生来自不同库的噬菌体颗粒（汤姆林森I和J的库和表库21），用于将生物淘选过程中使用制造商的说明（http://www.lifesciences.sourcebioscience.com/media/143421/tomlinsonij.pdf）。 注:我们使用多个库在我们的淘洗过程中，以增加可用抗体的多样性。 </li><li>简要地说，在200毫升的2×YT含有1％葡萄糖和氨苄青霉素100μg/ ml的振荡器上的三个库中培养细菌种群在37℃，直到OD 600为0.4。 </li><li>加KM13辅助噬菌体8×10 11至200毫升汤姆林森I和J库和VCSM13辅助噬菌体8×10 11至200毫升表库。在37℃下孵育文库样品用辅助噬菌体30分钟不摇动。 </li><li>离心机培养物在3000×g离心10分钟，并重新悬浮在200毫升的2×YT中含有0.1％的葡萄糖，氨苄青霉素100μg/ ml的卡那霉素和50微克/毫升细胞沉淀。 </li><li>孵育培养物O / N在30℃下振荡，然后旋转30分钟，在3300×g离心次日。 </li><li>在2.5 M氯化钠添加50毫升聚乙二醇（PEG）8000向200毫升从每个库上清液并在冰上孵育1小时。 PEG沉淀后，再旋转的解决方案，30分钟3300×g的。 </li><li>加1-2毫升磷酸盐缓冲盐水（PBS）中，以将细胞沉淀（取决于粒料的大小），并转移到微量离心管中。 </li><li>在11600×g下离心10分钟，然后在37℃偶尔混合孵育噬菌体颗粒在冰上1小时。收集上清液并储存于-80℃。 </li><li>之前冷冻，从三个库调小体积的噬菌体颗粒的另一微量离心管中，并滴定每个库分别用TG1细胞，以确定浓度。下面是滴度过程的一个库的描述。 <ol><li>成长TG1细胞OD 600 0.4。添加990微升PBS中，以6离心管。 </li><li>在第一管中添加10微升噬菌体颗粒。混合后，从管1个10微升并加入试管2.重复此步骤，其余4管。 </li><li>加入200μlTG1细胞对6管，并添加10微升六个噬菌体稀释这些管的。允许所述噬菌体颗粒为30分钟，与感染，在37℃无振摇。 </li><li>板100微升细胞对的Luria-BERTANI琼脂平板含有氨苄青霉素100微克/毫升（LBA）的。孵育O / N在37℃，然后使用的可见菌落的数量确定的PFU / ml的。 </li></ol></li></ol> 2.负生物淘选过程注意:避免噬菌体太多冻融整个生物淘洗过程。 ID被真正，最好是进行尽可能多的负淘选步骤可以在一天。在完成两轮针对每个负或正淘选后靶向节省一些噬菌体的情况下，污染的任何步骤是很重要的。 <ol><li> 12轮对牛血清白蛋白负平移的（BSA） <ol><li>加入4毫升PBS中1mg / ml的BSA溶液，以12免疫管孵育O / N在4℃。 </li><li>从上述三个库将1×10 12噬菌体颗粒（预留负平移过程中未来验证少量这种噬菌体混合物）。 </li><li>丢弃从所述第一管的BSA溶液并用PBS洗管2-3次，以除去任何未结合的BSA。 </li><li>添加组合噬菌体文库，以该第一管中，孵育在室温30分钟对旋转器，并收集未结合的噬菌体。 </li><li>重复步骤2.1.3与第二免疫管，并从步骤2.1.4收集的噬菌体添加到此我mmunotube。重复步骤2.1.4。 </li><li>与剩余的10管的BSA溶液与每一培养后收集的未结合的噬菌体重复步骤2.1.3-2.1.4。 </li><li>为了验证去除所有BSA的噬菌体粘合剂的使用AFM。步骤后添加10微升噬菌体从步骤2.1.2和10μl剩余的噬菌体的BSA 2.1.7分别至10μg/ ml的结合于切割的云母。在AFM过程的简单版本，在第8概述。 </li><li>它保存少量的噬菌体完成多轮负淘洗针对每个目标，以验证平移过程的成功之后是很重要的。 </li></ol></li><li> 10轮打击各种形式的α-突触核蛋白负的平移 注意:在这个项目中，我们要删除的噬菌体，可以结合蛋白如α-突触核蛋白（单体，二聚体，三聚体等 ），因此，我们将消除任何这些噬菌体的其他聚集形式（的目标是消除为许多交叉反应噬菌体越好）。让纯净的α突触核蛋白聚合至少7天，以便单体和低聚物的形式存在负平移。 <ol><li> 4，准备与免疫管聚合α-突触核蛋白在PBS 500微克/毫升和4个与免疫管聚合α-突触核蛋白100微克/毫升和孵化O / N在4℃。 </li><li>丢弃从与聚集的α-突触核蛋白的500微克/毫升的管中的一个的溶液，洗净，用PBS，并添加了12轮的负与BSA淘洗后收集的噬菌体文库。 </li><li>孵育管在室温30分钟对旋转器，并收集未结合的噬菌体。 </li><li>重复步骤2.2.3和2.2.4用于与聚集的α-突触核蛋白的500微克/毫升，然后用聚合α-突触核蛋白的每次使用收集步骤2.2.4之后未结合的噬菌体100微克/毫升的管中的剩余管中。 </li><li>使用噬菌体后负淘选的第一对聚集的α500微克/毫升，重复步骤2.1.8突触核蛋白和第八轮负淘选针对α-突触核蛋白的100微克/毫升后的噬菌体。 </li><li>为了消除对一切负面的粘合剂的α-突触核蛋白准备两个额外的免疫管与聚合α-突触核蛋白500微克/毫升，然后重复步骤2.2.2-2.2.4。 </li></ol></li><li> 10轮对健康的人脑组织匀浆负的平移<ol><li>加1×STEN缓冲液（50mM的Tris（pH值7.6），150 mM氯化钠，2mM EDTA的，0.2％的NP-40，0.2％BSA，20mM的PMSF和蛋白酶抑制剂鸡尾酒），以人脑组织28。均质在24000转1分钟，离心样品以3000rpm，并收集上清。 </li><li>准备2免疫管与2ml人脑组织的50微克/毫升在PBS中，8免疫管与人脑组织的10微克/毫升孵育O / N在4℃。 </li><li>重复步骤2.2.3-2.2.5先用含有人脑组织的50微克/毫升的管中，然后用含有人B的10微克/毫升雨组织使用针对α-突触核蛋白的负淘选后收集未结合的噬菌体。 </li><li>使用后的负淘选的第一对人脑组织的50微克/毫升和第十轮负淘选抗人脑组织后的噬菌体的噬菌体重复步骤2.1.8。 </li></ol></li><li> 8轮对健康免疫TDP-43负的平移<ol><li>免疫沉淀使用在第9描述的协议健康的人脑组织的健康TDP-43蛋白由于免疫沉淀蛋白质的量较低，开展这些负面轮淘洗用云母。 </li><li>切割8云母表面。 </li><li>到第一云母添加〜100微升的免疫沉淀的蛋白的5微克/毫升孵育在室温下5-10分钟。 </li><li>用0.1％的PBS吐温20（PBST）洗云母5次。 </li><li>添加〜100微升噬菌体对健康的脑组织的负面平移后孵育5-10分钟RT。 </li><li>在步骤2.4.5中途孵化，开展一步2.4.3使用第二云母。 </li><li>收集来自步骤2.4.5噬菌体并用0.1％PBST清洗云母5次。 </li><li>从步骤2.4.6洗云母和2.4.7增加噬菌体。 </li><li>重复步骤2.4.4至2.4.9，直到噬菌体对所有八个云母与免疫蛋白的负平移。用后的第一和八轮负淘选的噬菌体重复步骤2.1.7。 </li></ol></li><li> 2轮负平移对FTD免疫TDP-43 <ol><li>免疫沉淀TDP-43蛋白从与FTD个人的大脑。 </li><li>切割2云母表面。 </li><li>添加50微升FTD的10微克/毫升，TDP-43，以1云母。孵育5分钟，在室温。 </li><li>洗云母5次用0.1％PBST清洗。 </li><li>添加对健康TDP-43的负淘选后所收集的噬菌体为50μl。孵育5分钟。 </li><li>收集未结合的噬菌体。 </li><li> REPEATS步骤2.5.3至2.5.6与第二云母，并收集在2.5.6的未结合的噬菌体。 </li></ol></li></ol> 3.正对免疫TDP-43的个人与ALS平移<ol><li>免疫沉淀TDP-43蛋白从与ALS个人的大脑。切割3云母表面。加入50微升的ALS 10微克/毫升的TDP-43到1的云母。孵育5分钟，在室温。洗云母5次用0.1％PBST清洗。 </li><li>添加对健康TDP-43到云母负淘选后所收集的噬菌体为50μl。孵育5分钟。 </li><li>收集未结合的噬菌体。还收集使用三个洗脱方法结合的噬菌体。重要的是要保持所有收集的噬菌体在不同的这些步骤，平移是很重要的。 <ol><li>首先，添加50微升的胰蛋白酶（1mg / ml的在PBS中），以每云母孵育不超过10分钟以上。收集解决方案和保存。接下来，添加50微升的100mM三乙胺（TEA），以每云母孵育不超过10分钟以上。有限公司llect的溶液中，添加的1M的Tris-HCl缓冲液pH 7.4，并保存等量。 </li><li>第三，以0.4的OD 600添加50μl的TG1的直接向云母和温育30分钟，在37℃。另外在3.3.1至100μlTG1的以0.4的OD 600添加来自两个洗脱方法收集的噬菌体孵育30分钟，在37℃。 </li></ol></li><li>重复步骤3.1与第二云母。 </li><li>采取收集在3.3.2的未结合的噬菌体。使用第一云母的ALS TDP-43，并将其添加到第二云母患有ALS的TDP-43。孵育5分钟。 </li><li>重复所有步骤在3.3这些第二套云母。 </li><li>结合感染洗脱来自两个ALS TDP-43和云母板在LBA平板胰蛋白酶的噬菌体的TG1细胞。重复同样的过程在TEA和TG1洗脱从两ALS TDP-43的云母噬菌体，共三大板块。 注意:由于TDP-43参与两个ALS和FTD的，一些克隆中分离出的两轮针对ALS的TDP-43阳性淘选可以是特定于该靶，但有的也可以结合FTD。为了更好地隔离ALS特定克隆，采取后2轮对负平移收集到的未结合的噬菌体FTD TDP-43（因为这两轮负淘洗应该删除的是反应性FTD TDP-43的任何目标），并在使用这些噬菌体人更圆兑ALS TDP-43抗原云母积极平移（如3.3和3.4上述准备ALS TDP-43的云母）的。 </li><li>重复所有步骤在3.3与第三ALS云母板受感染的TG1细胞三个LBA板O / N在37℃。第二天计数的6 LBA板与潜在的ALS克隆的菌落数。应于三个LBA板与第三轮正淘选相比两轮正淘选后三个LBA板后的电势的ALS克隆更少菌落。 </li></ol> 4.培养和噬菌体产品潜在的阳性克隆离子对ALS具体TDP-43 <ol><li>从6平板中含有100微升的2×YT中有1％葡萄糖和氨苄青霉素100μg/ ml的在振荡器上在37℃CO / N 96孔板生长每一集落。 </li><li>第二天传递从含有从3 LBA平板上的克隆第三轮正淘选利用ALS TDP-43蛋白含有1ml的2×YT中有1％葡萄糖和氨苄青霉素100微克/毫升离心管后的孔中10微升。 </li><li>添加甘油到96孔板的所有培养物，以15％的终浓度，冷冻平板在-80℃下。允许1毫升培养中生长2-3小时，摇动，在37℃。 </li><li>加10的KM13辅助噬菌体9，并允许辅助噬菌体感染1小时，在37℃振荡。离心机在3000×g离心所述细胞10分钟，弃去上清液，并加入1 ml的2xYT的含0.1％葡萄糖氨苄青霉素，100微克/毫升和卡纳姆的50微克/毫升霉素。 </li><li>培养细胞O / N在30℃振荡。离心管在3300×g离心30分钟，将上清转移到新的微量离心管中，并微升PEG /氯化钠加入250到每个管中。 </li><li>冰上孵育管1小时。再次离心管在3300×g离心30分钟，弃去上清液，加入100μl的PBS中，以每管。 </li><li>冰上孵育管1小时。旋管10分钟，在11600×g离心，上清液转移到新管中。存储噬菌体在-80℃。 </li></ol> 5.测序潜在ALS的克隆<ol><li>重复步骤4.2和允许的文化成长O / N在37℃振荡。次日进行克隆的DNA质粒的Preps根据制造商的说明。使用适当的正向序列的克隆和反向引物。 </li></ol> 6.筛选潜在的阳性克隆针对ALS具体TDP-43使用间接ELISA <ol><li>加入100微升的ALS，FTD和健康的人脑组织的2.5微克/毫升到高结合ELISA板的每个潜在的ALS克隆的孔中。孵育所述板在37℃下1小时缓慢摇动。 </li><li>洗板3次，用0.1％PBST清洗。在PBS中加入200微升2％奶粉的井和孵化所述板在37℃下1小时缓慢摇动。 </li><li>重复洗涤步骤，然后添加100微升稀释在PBS中的至少1/100至孔的每个噬菌体。后的温育和洗涤步骤后，加入100微升的1 / 1,000抗M13 HRP到每个孔中。洗板和可视化使用的ELISA毫微微化学发光底物试剂盒。 </li></ol> 7.单链抗体生产和筛选使用间接ELISA <ol><li>成长HB 2151细胞OD 600 0.4。加入5微升不同的噬菌体至20微升的HB 2151的细胞。让噬菌体30分钟感染，在37°C时不晃动。分LBA板成网格等大约20个克隆可镀上的每个。 </li><li>加入5-10微升每个噬菌体感染细菌的板块和孵化O / N在37℃。第二天添加单独的菌落到1ml的2×YT中含0.1％葡萄糖和100氨苄青霉素微克/毫升和摇，在37℃下进行3-4小时，直到OD 600为0.6。 </li><li>加入1微升的1M异丙基（IPTG）至每个试管孵育O / N在30℃下振荡。旋管在11600×g离心10分钟，收获上清液用于ELISA测试。 </li><li>用于ELISA试验按照第6相同的方法，除了代替噬菌体在6.5添加未稀释的scFv和6.6添加的1 / 1,000 9E10 HRP加入100μl。 </li></ol> 8. AFM过程<ol><li>添加10-20微升靶抗原的对剥离的云母并孵育5-10分钟。用水洗涤云母5次。添加10-20微升噬菌体的云母并孵育5-10分钟。 </li><li>洗云母5倍再次WI次用水和允许云母风干。可视化噬菌体使用AFM 1约束力。执行使用AFM轻敲模式，在室温29在空气中的Nanoscope IIIa受体控制的AFM成像过程。硅AFM探针具有300kHz的共振频率和40 N / m的弹簧常数。使用3.05赫兹的扫描速率和512个样本/线的扫描分辨率。 </li><li>处理由背景的基本平坦的图像，以确保所有的颗粒大小为每个图像内相媲美。噬菌体宽度（未长度）可以从图像到图像由于其它颗粒尺寸和扫描区域有所不同，但它绝不会损害与否噬菌体​​的准确性与抗原结合。 </li><li>如果在AFM验证每组负淘选噬菌体是可见的云母，直到没有噬菌体在进行下一组负淘选之前检测到执行加法轮。 </li></ol> 9.免疫沉淀靶抗原<ol><li>为了isolatE中的靶抗原（TDP-43）中，添加250均质脑组织（从运动皮层），加入100μl的1/100稀释的抗TDP 43多克隆抗体和1x STEN缓冲液至700微升总体积到Eppendorf微克管28。 </li><li>孵育O / N在4℃旋转器上。洗A / G琼脂糖珠与1X STEN缓冲器2-3倍，加25毫升的混合组织。孵育在4℃下1.5小时在旋转器上，然后离心以2,000-4,000转速1分钟。 </li><li>去除上清，并用1×STEN缓冲液和5次，用PBS洗涤沉淀一次。添加40-60微升1×STEN缓冲器向沉淀并煮沸5分钟。样品后凉爽，离心收集上清。使用SDS-PAGE和Western印迹验证免疫过程30成功。 </li></ol> 10点印迹分析<ol><li>切断硝基纤维素纸成小的矩形为将要测试的每个克隆。点2微升ALS，女的TD和健康人的脑组织样本对硝酸纤维素纸，让它完全干燥。 </li><li>加入1-2毫升的5％奶粉的PBS中，并让它摇动1小时，在室温。加入1/100稀释每个噬菌体1毫升，让它摇O / N在4℃。每10分钟，用0.​​5％PBST洗膜3次。 </li><li>加入1毫升的1 / 1,000稀释抗M13的HRP，并让它摇动1小时，在室温。重复清洗过程，用西方化学发光的解决方案可视化。 </li></ol>

<ol>
	<li>Hedieh, B., Sharareh, E., Philip, S., Michael, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+recombinant+antibodies+against+specific+protein+morphologies+using+atomic+force+microscopy+and+phage+display+technologies.">Isolating recombinant antibodies against specific protein morphologies using atomic force microscopy and phage display technologies.</a> Protein Engineering Design and Selection. 19, 497-502 (2006).</li><li>Emadi, S., Barkhordarian, H., Wang, M. S., Schulz, P., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+Human+Single+Chain+Antibody+Fragment+Against+Oligomeric+&#945;-Synuclein+that+Inhibits+Aggregation+and+Prevents+&#945;-Synuclein-induced+Toxicity.">Isolation of a Human Single Chain Antibody Fragment Against Oligomeric &#945;-Synuclein that Inhibits Aggregation and Prevents &#945;-Synuclein-induced Toxicity.</a> Journal of Molecular Biology. 368, 1132-1144 (2007).</li><li>Emadi, S., Kasturirangan, S., Wang, M. S., Schulz, P., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+Morphologically+Distinct+Oligomeric+Forms+of+&#945;-Synuclein.">Detecting Morphologically Distinct Oligomeric Forms of &#945;-Synuclein.</a> Journal of Biological Chemistry. 284, 11048-11058 (2009).</li><li>Kasturirangan, S., 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=Nanobody+specific+for+oligomeric+beta-amyloid+stabilizes+nontoxic+form.">Nanobody specific for oligomeric beta-amyloid stabilizes nontoxic form.</a> Neurobiology of Aging. 33, 1320-1328 (2012).</li><li>Kasturirangan, S., 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=Isolation+and+characterization+of+antibody+fragments+selective+for+specific+protein+morphologies+from+nanogram+antigen+samples.">Isolation and characterization of antibody fragments selective for specific protein morphologies from nanogram antigen samples.</a> Biotechnology Progress. 29, 463-471 (2013).</li><li>Zameer, A., Kasturirangan, S., Emadi, S., Nimmagadda, S. V., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-oligomeric+A&#946;+Single-chain+Variable+Domain+Antibody+Blocks+A&#946;-induced+Toxicity+Against+Human+Neuroblastoma+Cells.">Anti-oligomeric A&#946; Single-chain Variable Domain Antibody Blocks A&#946;-induced Toxicity Against Human Neuroblastoma Cells.</a> Journal of Molecular Biology. 384, 917-928 (2008).</li><li>Boddapati, S., Levites, Y., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+&#946;-Secretase+Activity+in+Alzheimer's+Disease+Cell+Models+with+Single-Chain+Antibodies+Specifically+Targeting+APP.">Inhibiting &#946;-Secretase Activity in Alzheimer's Disease Cell Models with Single-Chain Antibodies Specifically Targeting APP.</a> Journal of Molecular Biology. 405, 436-447 (2011).</li><li>Boddapati, S., Levites, Y., Suryadi, V., Kasturirangan, S., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bispecific+Tandem+Single+Chain+Antibody+Simultaneously+Inhibits+&#946;-Secretase+and+Promotes+&#945;-Secretase+Processing+of+A&#946;PP.">Bispecific Tandem Single Chain Antibody Simultaneously Inhibits &#946;-Secretase and Promotes &#945;-Secretase Processing of A&#946;PP.</a> Journal of Alzheimer's Disease. 28, 961-969 (2012).</li><li>Zhou, C., Emadi, S., Sierks, M. R., Messer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Human+Single-Chain+Fv+Intrabody+Blocks+Aberrant+Cellular+Effects+of+Overexpressed+[alpha]-Synuclein.">A Human Single-Chain Fv Intrabody Blocks Aberrant Cellular Effects of Overexpressed [alpha]-Synuclein.</a> Mol Ther. 10, 1023-1031 (2004).</li><li>Vanden Broeck, L., Callaerts, P., Dermaut, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43-mediated+neurodegeneration:+towards+a+loss-of-function+hypothesis.">TDP-43-mediated neurodegeneration: towards a loss-of-function hypothesis.</a> Trends in Molecular Medicine. 20, 66-71 (2014).</li><li>Akamatsu, M., 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+unique+mouse+model+for+investigating+the+properties+of+amyotrophic+lateral+sclerosis-associated+protein+TDP-43,+by+in+utero+electroporation.">A unique mouse model for investigating the properties of amyotrophic lateral sclerosis-associated protein TDP-43, by in utero electroporation.</a> Neuroscience Research. 77, 234-241 (2013).</li><li>Keage, H. A., 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=TDP-43+in+the+Population:+Prevalence+and+Associations+with+Dementia+and+Age.">TDP-43 in the Population: Prevalence and Associations with Dementia and Age.</a> Journal of Alzheimer's Disease. 42, 641-650 (2014).</li><li>Honda, D., 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+ALS/FTLD-related+RNA-binding+proteins+TDP-43+and+FUS+have+common+downstream+RNA+targets+in+cortical+neurons.">The ALS/FTLD-related RNA-binding proteins TDP-43 and FUS have common downstream RNA targets in cortical neurons.</a> FEBS Open Bio. 4, 1-10 (2014).</li><li>Baloh, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43:+the+relationship+between+protein+aggregation+and+neurodegeneration+in+amyotrophic+lateral+sclerosis+and+frontotemporal+lobar+degeneration.">TDP-43: the relationship between protein aggregation and neurodegeneration in amyotrophic lateral sclerosis and frontotemporal lobar degeneration.</a> FEBS Journal. 278, 3539-3549 (2011).</li><li>Ling, S. -. C., Polymenidou, M., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Converging Mechanisms in ALS and FTD: Disrupted RNA and Protein. 79, 416-438 (2013).</li><li>Sasaki, S., Takeda, T., Shibata, N., Kobayashi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+subcellular+localization+of+TDP-43+immunoreactivity+in+the+anterior+horns+in+sporadic+amyotrophic+lateral+sclerosis.">Alterations in subcellular localization of TDP-43 immunoreactivity in the anterior horns in sporadic amyotrophic lateral sclerosis.</a> Neuroscience Letters. 478, 72-76 (2010).</li><li>Robertson, J., 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=Lack+of+TDP-43+abnormalities+in+mutant+SOD1+transgenic+mice+shows+disparity+with+ALS.">Lack of TDP-43 abnormalities in mutant SOD1 transgenic mice shows disparity with ALS.</a> Neuroscience Letters. 420, 128-132 (2007).</li><li>Shan, X., Vocadlo, D., Krieger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mislocalization+of+TDP-43+in+the+G93A+mutant+SOD1+transgenic+mouse+model+of+ALS.">Mislocalization of TDP-43 in the G93A mutant SOD1 transgenic mouse model of ALS.</a> Neuroscience Letters. 458, 70-74 (2009).</li><li>Yamashita, T., Hideyama, T., Teramoto, S., Kwak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+abnormal+processing+of+TDP-43+is+not+an+upstream+event+of+reduced+ADAR2+activity+in+ALS+motor+neurons.">The abnormal processing of TDP-43 is not an upstream event of reduced ADAR2 activity in ALS motor neurons.</a> Neuroscience Research. 73, 153-160 (2012).</li><li>Dong, H., 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=Curcumin+abolishes+mutant+TDP-43+induced+excitability+in+a+motoneuron-like+cellular+model+of+ALS.">Curcumin abolishes mutant TDP-43 induced excitability in a motoneuron-like cellular model of ALS.</a> Neuroscience. 272, 141-153 (2014).</li><li>Sheets, M. D., 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=Efficient+construction+of+a+large+nonimmune+phage+antibody+library:+The+production+of+high-affinity+human+single-chain+antibodies+to+protein+antigens.">Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens.</a> Proceedings of the National Academy of Sciences. 95, 6157-6162 (1998).</li><li>Hairul Bahara, N. H., 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=Phage+display+antibodies+for+diagnostic+applications.">Phage display antibodies for diagnostic applications.</a> Biologicals. 41, 209-216 (2013).</li><li>Azzazy, H. M. E., Highsmith, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+display+technology:+clinical+applications+and+recent+innovations.">Phage display technology: clinical applications and recent innovations.</a> Clinical Biochemistry. 35, 425-445 (2002).</li><li>Zhang, X., 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=Rapid+isolation+of+single-chain+antibodies+from+a+human+synthetic+phage+display+library+for+detection+of+Bacillus+thuringiensis+(Bt).">Rapid isolation of single-chain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis (Bt).</a> Cry1B toxin. Ecotoxicology and Environmental Safety. 81, 84-90 (2012).</li><li>Liu, H., 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=Selection+and+characterization+of+single-chain+recombinant+antibodies+against+spring+viraemia+of+carp+virus+from+mouse+phage+display+library.">Selection and characterization of single-chain recombinant antibodies against spring viraemia of carp virus from mouse phage display library.</a> Journal of Virological Methods. 194, 178-184 (2013).</li><li>Cukkemane, N., Bikker, F. J., Nazmi, K., Brand, H. S., Veerman, E. C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+salivary-pellicle-binding+peptide+by+phage+display.">Identification and characterization of a salivary-pellicle-binding peptide by phage display.</a> Archives of Oral Biology. 59, 448-454 (2014).</li><li>Adamson, C. S., 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=Novel+single+chain+antibodies+to+the+prion+protein+identified+by+phage+display.">Novel single chain antibodies to the prion protein identified by phage display.</a> Virology. 358, 166-177 (2007).</li><li>Hebron, M. 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=Parkin+Ubiquitinates+Tar-DNA+Binding+Protein-43+(TDP-43)+and+Promotes+Its+Cytosolic+Accumulation+via+Interaction+with+Histone+Deacetylase+6+(HDAC6).">Parkin Ubiquitinates Tar-DNA Binding Protein-43 (TDP-43) and Promotes Its Cytosolic Accumulation via Interaction with Histone Deacetylase 6 (HDAC6).</a> Journal of Biological Chemistry. 288, 4103-4115 (2013).</li><li>Wang, M. S., Zameer, A., Emadi, S., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+Antibody+Specificity+to+Different+Protein+Morphologies+by+AFM.">Characterizing Antibody Specificity to Different Protein Morphologies by AFM.</a> Langmuir. 25, 912-918 (2008).</li><li>Williams, S., Sakic, B., Hoffman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+brain-reactive+autoantibodies+and+behavioral+deficits+in+the+MRL+model+of+CNS+lupus.">Circulating brain-reactive autoantibodies and behavioral deficits in the MRL model of CNS lupus.</a> Journal of Neuroimmunology. 218, 73-82 (2010).</li><li>Jo&#324;czyk, E., K&#322;ak, M., Mi&#281;dzybrodzki, R., G&#243;rski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+external+factors+on+bacteriophages&#8212;review.">The influence of external factors on bacteriophages&#8212;review.</a> Folia Microbiol. 56, 191-200 (2011).</li><li>Hammers, C. M., Stanley, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+Phage+Display:+Technique+and+Applications.">Antibody Phage Display: Technique and Applications.</a> J Invest Dermatol. 134, e17 (2014).</li></ol>

The authors have nothing to disclose.

Protein variants have been shown to be involved in the progression of many neurodegenerative diseases such as Alzheimer&#8217;s, Parkinson&#8217;s, ALS and FTD1,2,3,4,5,6,7,8,9,10,11. Isolation of antibodies that can selectively recognize these different protein variant targets can be effective reagents to study, diagnose and potentially treat such ailments. To generate such variant specific antibodies we have developed a novel biopanning process that utilizes atomic force microscopy to monitor the progress ...

蛋白质变体的存在已被认为在许多疾病，包括神经变性疾病如阿尔茨海默氏症，帕金森氏，ALS和额颞痴呆（FTD）1,2,3,4,5,6,7,8,9的进展的一个因素，10,11。蛋白质的β-淀粉样蛋白和α-突触核蛋白的寡聚形式被认为是负责阿尔茨海默氏症和帕金森氏分别2,3,4,5有毒物种。在TAR DNA结合蛋白43（TDP-43）的聚集已与ALS和FTD 12,13,14。因此试剂如能够选择性靶向不同的蛋白质的变体可以是有力工具作为诊断标志物和潜在的治疗剂的抗体。在这项研究中，我们专注于开发的试剂选择性结合的TDP-43蛋白牵连在ALS的变体，但在本文中概述的技术应当适用于试剂的隔离针对广泛PROT的EIN变种。 TDP-43的细胞浆聚合已被确定为在ALS 15,16,17,18,19病理特征。典型地，TDP-43是在从正常个体的所有细胞的细胞核中，虽然它趋向于胞质溶胶和细胞核15,17之间移动。 TDP-43。然而，在ALS聚集形式中选择的神经元的细胞质和神经胶质与在细胞核中的疾病进展16,20暗示TDP-43的从细胞核向细胞质的移动发现低浓度的被检测到。而TDP-43的聚合中的大多数的ALS病例发现，它不自1％的总的ALS病例（或15％的家族性ALS例-20％）-2％被链接到在所述突变占所有病例超氧化物歧化酶1（SOD1）基因15,17。由于TDP-43在ALS的情况下，绝大多数的重要作用，在这里，我们专注于开发基于抗体的试剂，可以选择性结合TDP-43的变体是目前在利用我们的新型AFM基于生物淘洗技术人力ALS脑组织。 首先，我们需要的抗体结合结构域的不同剧目。我们结合三个不同的噬菌体展示单链可变区抗体片段（scFv的）库，（汤姆林森I和J和表库21）。所述平移过程分为阴性和阳性平移相。来自文库的噬菌体首先经受负淘选过程噬菌体其间反应到多个脱靶抗原被排除。在完成每一轮负淘选针对每个脱靶抗原后，该过程是通过原子力显微镜成像监测，以确保所有的噬菌体结合的脱靶抗原已被删除。只有原子力显微镜成像，所有反应噬菌体被删除核实后，我们进入到下一个目标。为了隔离对TDP-43的变种牵连ALS试剂，我们利用一系列的负面平移抗原:<html> 1）的BSA以除去噬菌体结合弱的或非特异性的蛋白质; 2）聚合α-突触核蛋白除去噬菌体结合到聚集的蛋白质的一般结构元件; 3）人脑组织匀浆以除去噬菌体结合于任何蛋白或本健康人脑组织验尸样品中其它组分; 4）免疫TDP-43从健康人的大脑，除去噬菌体结合与健康人的大脑相关的所有TDP-43表格;和5）免疫沉淀TDP-43从FTD脑匀浆中分离除去噬菌体结合与非ALS的病理相关联的TDP-43变体。去除所有的噬菌体的反应性的所有脱靶抗原后，我们接着就在此期间，该结合目的抗原的抗体片段被隔离的正淘选相，在这种情况下，TDP-43从人类ALS的脑组织中免疫沉淀。这些分离的抗体可以是反应性的TDP-43的凝集或修饰的形式。 "&gt;传统噬菌体生物淘选主要集中在正淘选阶段22,23。一般感兴趣的靶被固定，所述噬菌体文库并将结合的噬菌体洗脱。噬菌体，然后放大并再次加入到目标，这扩增和培养过程通常重复几次，以增加的阳性结合噬菌体的百分比。尽管这种方法的变体已被广泛使用，以隔离抗体试剂对多种靶抗原的，它们一般都需要大量的纯化的靶抗原24,25,26的， 27，而我们的过程只需要微量的靶抗原的量。这里所描述的协议可以用于分离选择性结合靶抗原存在于非常低的浓度，而不需要纯化和平移可以直接针对进行试剂目前复杂的组织样本中的抗原，利用详尽的负面平移协议的验证用AFM确保克隆中分离针对抗原阳性应选择性地结合靶即使在不进行纯化或富集。 Kasturirangan和他的同事（2003年）都进行了类似的消极和积极的生物淘洗过程隔离的抗体反应来使用目标5纳克的浓度低聚β-淀粉样蛋白。在这里，我们在这个过程中展开，使试剂选择性结合疾病特异性蛋白变体直接从人组织样品的产生。在未来的研究中，我们打算不仅要进一步调查此隔离试剂的诊断价值，而且评估其治疗的相关治疗ALS。 总的来说，我们的新颖的AFM基于生物淘选技术应当适用于任何特定疾病的蛋白质变体在任何生物材料的分离而无需蛋白质纯化或修饰，即使当靶抗原concentratioNS是非常低的。

<table border="0" cellpadding="0" cellspacing="0" width="940">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Reagent/ Equipment</td>
			<td style="width:287px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:212px;">Comments/Description</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:323px;">Tomlinson I and J Libraries</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sheets Library</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2xYT</td>
			<td style="width:287px;">BD Sciences</td>
			<td style="width:119px;">244020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Glucose</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0188-2.5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Ampicillin</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0339-25G</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">KM13 Helper Phage</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Kanamycin</td>
			<td style="width:287px;">OmniPur</td>
			<td style="width:119px;">5880</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Polyethylene Glycol 8000</td>
			<td style="width:287px;">OmniPur</td>
			<td style="width:119px;">6510</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium Chloride</td>
			<td style="width:287px;">Macron</td>
			<td style="width:119px;">7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium Phosphate Dibasic</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0404-1KG</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Potassium Chloride</td>
			<td style="width:287px;">EMD</td>
			<td style="width:119px;">PX1405-1</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Potassium Phosphate Monobasic</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0781-500G</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">TG1 Cells</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria-Bertani Agar</td>
			<td style="width:287px;">EMD</td>
			<td style="width:119px;">1.10283.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Bovine Serum Albumin</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0332-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">STEN buffer</td>
			<td style="width:287px;">Crystalgen Inc.</td>
			<td style="width:119px;">33429775</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Immunotubes</td>
			<td style="width:287px;">Thermo Scientific</td>
			<td style="width:119px;">470319</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Mica</td>
			<td style="width:287px;">Spruce Pine Mica</td>
			<td style="width:119px;">24365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Tween 20</td>
			<td style="width:287px;">EMD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Trypsin</td>
			<td style="width:287px;">Sigma</td>
			<td style="width:119px;">T-0303</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triethylamine</td>
			<td style="width:287px;">Sigma</td>
			<td style="width:119px;">T-0886</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Glycerol</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0854-1L</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DNA Plasmid Prep Kit</td>
			<td style="width:287px;">qiagen</td>
			<td style="width:119px;">27106</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Non-Fat Milk Powder</td>
			<td style="width:287px;">Carnation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">96-Well High Binding ELISA Plate</td>
			<td style="width:287px;">Costar</td>
			<td style="width:119px;">3590</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-M13 HRP</td>
			<td style="width:287px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">27-9421-01</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:323px;">ELISA Femto Chemiluminescence Substrate Kit</td>
			<td style="width:287px;">Thermo Scientific</td>
			<td style="width:119px;">37074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-TDP 43 Polyclonal Antibody</td>
			<td style="width:287px;">ProteinTech</td>
			<td style="width:119px;">10782-2-AP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">A/G Agarose Beads</td>
			<td style="width:287px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">HB 2151 Cells</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropylthiogalactoside</td>
			<td style="width:287px;">Teknova</td>
			<td style="width:119px;">13325</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">9e10 HRP</td>
			<td style="width:287px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Nitrocellulose Membrane</td>
			<td style="width:287px;">Biorad</td>
			<td style="width:119px;">162-0115</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Centrifuge</td>
			<td style="width:287px;">Thermo Scientific</td>
			<td style="width:119px;">Sorvall RC 6+</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanoscope IIIa Atomic Force Microscope</td>
			<td style="width:287px;">Veeco</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AFM Probes</td>
			<td style="width:287px;">VistaProbes</td>
			<td style="width:119px;">T300R-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

novel atomic force microscopy based biopanning for isolation of morphology specific reagents against tdp-43 variants in amyotrophic lateral sclerosis

因为蛋白变体中的许多疾病，包括TDP-43在肌萎缩性脊髓侧索硬化症（ALS），α-突触核蛋白中发挥关键作用帕金森氏病和β-淀粉样蛋白和tau蛋白在阿尔茨海默氏病，这是极为重要的发展形态特异性试剂，可以选择性地靶向这些疾病特异性蛋白变体来研究疾病的病理和潜在的诊断和治疗应用这些变体的作用。我们已经开发出新颖的原子力显微镜（AFM）基于生物淘选技术，使试剂选择性识别特定疾病的蛋白质变体的分离。有参与的过程中，负和正淘选相位的两个主要阶段。在负相淘选，噬菌体是反应性的脱靶抗原，通过多轮利用一系列精心挑选的脱靶抗原消减淘选被消除。否定一个主要特征平移相是利用原子力显微镜成像以监测过程，并确认所有不希望的噬菌体颗粒被除去。用于正淘选阶段中，感兴趣的靶抗原被固定在云母表面和结合的噬菌体洗脱，并筛选以鉴定噬菌体选择性结合靶抗原。靶蛋白变体不需要进行纯化提供适当的阴性对照摇动已被使用。即使靶蛋白变体是唯一存在的非常低的浓度在复杂的生物材料可以在正淘选步骤中使用。通过应用这种技术，我们收购了抗体TDP-43是在人类ALS脑组织选择性地发现蛋白变体。我们预计，这个协议应适用于生成试剂选择性结合蛋白变体中存在的多种不同的生物过程和疾病。

在图1中，示意性说明了负淘选过程，其中我们除去噬菌体使用免疫管我们的文库结合脱靶抗原。我们最初开始与BSA，因为这是一种常见的封闭剂和任何噬菌体，将非特异性反应与此目标将是有问题的未来免疫测定。接下来，我们除去粘结剂以聚集的α-突触核蛋白消除噬菌体是与聚集蛋白（ 即，抗体，这将是交叉反应性的，以聚集的α-突触核蛋白，TDP-43，Abeta的等 ）的通...

Watch this Scientific Journal Video about Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis at JoVE.

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

新型原子力显微镜基于生物淘选的形态特殊试剂对TDP-43变型肌萎缩侧索硬化症隔离

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

novel-atomic-force-microscopy-based-biopanning-for-isolation

Research

JoVE Journal

Bioengineering

8.7K 次观看.  Arizona State University. Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

视频: Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

重组DNA和蛋白质复合物，用原子力显微镜的可视化

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

科研

生物工程

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

量化的内皮细胞糖复合物的力学性能，用原子力显微镜

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

细菌表面电位测量用开尔文探针力显微镜

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved oxygen concentration). Nevertheless, the morphology of cells can be a suitable indicator for optimal conditions, since it changes with dependence on the growth state, product accumulation and cell stress. Furthermore, the single-cell size distribution provides not only information about the cultivation conditions, but also about the population heterogeneity. To gain such information, a photo-optical in situ microscopy device1 was developed to enable the monitoring of the single-cell size distribution directly in the cell suspension in bioreactors. An automated image analysis is coupled to the microscopy based on a neural network model, which is trained with user-annotated images. Several parameters, which are gained from the captures of the microscope, are correlated to process relevant features of the cells, like their metabolic activity. Until now, the presented in situ microscopy probe series was applied to measure the pellet size in filamentous fungi suspensions. It was used to distinguish the single-cell size in microalgae cultivation and relate it to lipid accumulation. The shape of cellular particles was related to budding in yeast cultures. The microscopy analysis can be generally split into three steps: (i) image acquisition, (ii) particle identification, and (iii) data analysis, respectively. All steps have to be adapted to the organism, and therefore specific annotated information is required in order to achieve reliable results. The ability to monitor changes in cell morphology directly in line or on line (in a by-pass) enables real-time values for monitoring and control, in process development as well as in production scale. If the off line data correlates with the real-time data, the current tedious off line measurements with unknown influences on the cell size become needless.

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

在原位生物过程单细胞形态实时测定的显微镜

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

原子力显微镜在早期骨关节炎检测中的应用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

数百个 C.白化病 细胞生物原子力显微镜测量自动化

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...