Name: novel atomic force microscopy based biopanning for isolation of morphology specific reagents against tdp-43 variants in amyotrophic lateral sclerosis
Uploaded: 2015-02-12T15:00:00.000+00:00
Duration: 13 min 31 s
Description: 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.

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>

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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 of each step. Previous work using a similar negative and positive biopanning combination resulted in a clone that binds oligomeric beta-amyloid5. Here we extend these studies to demonstrate that we can efficiently generate reagents that selectively bind disease-specific protein variants present in human brain tissue samples. We isolated morphology specific reagents that selectively recognize TDP-43 variants uniquely present in human ALS brain tissue13,14. Future studies necessitate characterization of the TDP-43 variants being targeted (whether aggregated or modified).
The key steps to ensuring that we can obtain phages specific to any protein variant target during the positive panning phase is to confirm that the negative panning steps have removed all potential cross-reacting phage clones. We utilized AFM imaging to verify that essentially all phage clones binding off-target antigens have been removed during each of the rounds of negative panning. Since a high percentage of the phage recovered from the positive binding are then selective for the desired target, only minimal amounts of the target antigen is needed for screening. 
It is essential to perform any of the panning steps where the phages will be exposed to the external environment in a biosafety cabinet to prevent contamination by other phage. To guard against this event, it is important to save a small aliquot of phage after each panning round. If contamination is discovered, a phage aliquot from a previous round can then be used to continue the process. Freezing and thawing the phages several times may be harmful to stability and so it is beneficial to perform many of the negative panning steps continuously31. Because the panning process does not utilize phage amplification steps after each round of positive panning32 the process is less likely to isolate empty or incomplete phage sequences. 
The AFM based panning process generates a number of potential protein variant specific clones after the positive panning step. To reduce the number of potential clones, we tested each clone for its ability to distinguish ALS from other diseased and healthy human brain tissue samples. The AFM based biopanning process described here represents a powerful technique that can be used to isolate reagents that selectively recognize disease-specific protein variants directly from complex human samples. The target protein variants can be present at very low concentrations, and there is no need to purify, modify or concentrate the target antigen.

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><tbody><tr><td>Tomlinson I and J Libraries</td><td>MRC (Cambridge, England)</td><td></td><td></td></tr><tr><td>Sheets Library</td><td>MRC (Cambridge, England)</td><td></td><td></td></tr><tr><td>2xYT</td><td>BD Sciences</td><td>244020</td><td></td></tr><tr><td>Glucose</td><td>Amresco</td><td>0188-2.5KG</td><td></td></tr><tr><td>Ampicillin</td><td>Amresco</td><td>0339-25G</td><td>Irritant</td></tr><tr><td>KM13 Helper Phage</td><td>MRC (Cambridge, England)</td><td></td><td></td></tr><tr><td>Kanamycin</td><td>OmniPur</td><td>5880</td><td>Irritant</td></tr><tr><td>Polyethylene Glycol 8000</td><td>OmniPur</td><td>6510</td><td>Irritant</td></tr><tr><td>Sodium Chloride</td><td>Macron</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Amresco</td><td>0404-1KG</td><td>Irritant</td></tr><tr><td>Potassium Chloride</td><td>EMD</td><td>PX1405-1</td><td>Irritant</td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Amresco</td><td>0781-500G</td><td>Irritant</td></tr><tr><td>TG1 Cells</td><td>MRC (Cambridge, England)</td><td></td><td></td></tr><tr><td>Luria-Bertani Agar</td><td>EMD</td><td>1.10283.0500</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Amresco</td><td>0332-100G</td><td></td></tr><tr><td>STEN buffer</td><td>Crystalgen Inc.</td><td>33429775</td><td></td></tr><tr><td>Immunotubes</td><td>Thermo Scientific</td><td>470319</td><td></td></tr><tr><td>Mica</td><td>Spruce Pine Mica</td><td>24365</td><td></td></tr><tr><td>Tween 20</td><td>EMD</td><td></td><td></td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T-0303</td><td>Irritant</td></tr><tr><td>Triethylamine</td><td>Sigma</td><td>T-0886</td><td>Flammable</td></tr><tr><td>Glycerol</td><td>Amresco</td><td>0854-1L</td><td>Irritant</td></tr><tr><td>DNA Plasmid Prep Kit</td><td>qiagen</td><td>27106</td><td>Irritant</td></tr><tr><td>Non-Fat Milk Powder</td><td>Carnation</td><td></td><td></td></tr><tr><td>96-Well High Binding ELISA Plate</td><td>Costar</td><td>3590</td><td></td></tr><tr><td>Anti-M13 HRP</td><td>GE Healthcare Life Sciences</td><td>27-9421-01</td><td></td></tr><tr><td>ELISA Femto Chemiluminescence Substrate Kit</td><td>Thermo Scientific</td><td>37074</td><td></td></tr><tr><td>Anti-TDP 43 Polyclonal Antibody</td><td>ProteinTech</td><td>10782-2-AP</td><td></td></tr><tr><td>A/G Agarose Beads</td><td>Santa Cruz Biotechnology</td><td>sc-2003</td><td></td></tr><tr><td>HB 2151 Cells</td><td>MRC (Cambridge, England)</td><td></td><td></td></tr><tr><td>Isopropylthiogalactoside</td><td>Teknova</td><td>13325</td><td></td></tr><tr><td>9e10 HRP</td><td>Santa Cruz Biotechnology</td><td>sc-40</td><td></td></tr><tr><td>Nitrocellulose Membrane</td><td>Biorad</td><td>162-0115</td><td>Flammable</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>Sorvall RC 6+</td><td></td></tr><tr><td>Nanoscope IIIa Atomic Force Microscope</td><td>Veeco</td><td></td><td></td></tr><tr><td>AFM Probes</td><td>VistaProbes</td><td>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 ）的通用结构的反应性。 AFM结果表明噬菌体后1轮负平移对聚合α-突触核蛋白（ 图2A）结合后8轮（ 图2B）没有任何约束力。然后，我们对健康人脑组织负摇除去噬菌体结合存在于健康人的脑匀浆的众多的抗原。 图2C </stron克&gt;表明噬菌体后1轮淘选负结合于健康组织和无噬菌体离开后10轮的负淘选（ 图2D）的结合。由于健康和FTD免疫TDP-43的蛋白质可用于淘选的量为低，使用云母而非免疫管使用较少的体积，因而降低了总耗电量蛋白（ 图3）。经过8轮负淘洗对健康的免疫TDP-43，噬菌体被划分。一半的噬菌体花费在两回合对FTD TDP-43的负平移的。其目的是消除任何FTD TDP-43的反应性的克隆（由于TDP-43参与FTD），同时保留了任何潜在的ALS TDP-43特异性克隆。 用于正淘选部（ 图4）中，我们也使用云母作为底物，以减少材料的使用。我们使用的未结合的噬菌体对ALS TDP-43获得任何FTD TDP-43的负面平移后ALS具体的克隆。我们还积极对ALS，因为我们使用的未结合的噬菌体从对FTD TDP-43的负面平移后没有获得克隆事件平移TDP-43的两倍。整个平移过程结束后，我们的三个洗脱方法产生154个克隆对ALS TDP-43的两个正平移（克隆，可能是反应性ALS和FTD）和45个潜在的ALS特异性克隆（使用从FTD TDP-的未结合的噬菌体43负平移）。 为了进一步减少45潜在的ALS特异性克隆，所述克隆测序，并只没有任何终止密码子的克隆被进一步考虑，除其中出现了两次一个克隆。这给我们留下了23潜在ALS具体的克隆。制备两个噬菌体和可溶性scFv与这些克隆后，它们在间接ELISA中对特异性的ALS的组织进行了测试。几乎所有的噬菌体显示偏爱的ALS组织超过健康组织（ 图5）。类似的结果是OBTained比较噬菌体结合到ALS到FTD组织（ 图6）时。对于未来的研究很重要的是这些克隆表达的功能性的scFv高产率，所以我们所产生的不同的scFv的小批量，并进行了类似的间接ELISA中。的单链抗体结合的ALS组织到两个健康（ 图7）和FTD组织（ 图8）的比较显示，再次选择在几乎所有的克隆结合的ALS组织。 我们使用的另一种免疫测定（点印迹），以进一步验证结合ALS组织并且还确定哪些克隆在其它免疫反应的应用。克隆2A结合ALS组织示（ 图9）。所有这些克隆中显示出有前景的结果的噬菌体和scFv的ELISA的任一个或两个。这些结果证实，我们的AFM基于生物淘选过程是一个非常强大的技术，可用于生成试剂选择性结合疾病特异性蛋白瓦里直接从来源复杂的蚂蚁。 <img alt="图1" src="/files/ftp_upload/52584/52584fig1highres.jpg" /> 图1.负生物淘选过程利用免疫管。示意图展示了负生物淘选过程。噬菌体是活性蛋白质，如BSA，聚集的α-突触核蛋白和健康的脑组织正在使用免疫管取出。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52584/52584fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52584/52584fig2highres.jpg" /> 图2中的负摇摄结果确认。AFM成像用于监视对各个目标的负淘选步骤，以确保所有的反应性的噬菌体将被删除。在这里，我们表现出一定的AFM结果展示噬菌体前后负淘洗后绑定的水平。（A）噬菌体结合第一轮负淘选后可以检测到聚合的α突触核蛋白颗粒，但（B）没有噬菌体都是经过8轮负淘洗可见。（C ）后第一轮淘选的负对健康组织的噬菌体结合是明显的，但经过10轮负淘选（D）的无结合被检测。所有图像都5微米。在AFM图像的白色大结构通常是由于存在于噬菌体生产过程中的PEG沉淀步骤缓冲区或残留PEG盐。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52584/52584fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52584/52584fig3highres.jpg" /> 图3.负Biopann荷兰国际集团工艺利用云母，云母使用示意图示范额外的负面生物淘洗。由于有限的样本云母表面采用先消除粘合剂健康，然后FTD免疫TDP-43。在开始的两回合对FTD TDP-43的负淘洗，噬菌体被劈成两半（在ALS的TDP-43的独家噬菌体在积极的平移阶段不成功隔离的情况下）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52584/52584fig3large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图4" src="/files/ftp_upload/52584/52584fig4highres.jpg" /> 图4. ALS正生物淘选过程。示意图表明了ALS积极的生物淘洗过程。针对FTD TDP-43的负淘选后的未结合的噬菌体用在轮正淘选针对ALS的TDP-43，以洗脱更多的ALSTDP-43特异性克隆。此外，两回合对ALS TDP-43阳性平移都进行了使用云母表面和对健康的免疫TDP-43（绑定的噬菌体洗脱，因为这些噬菌体不应该结合健康的TDP-43，但一些负面淘洗后绑定噬菌体可以是交叉反应既ALS和FTD）。三种方法洗脱使用（胰蛋白酶，TEA和TG1细胞），以确保所有结合的噬菌体被删除。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52584/52584fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52584/52584fig5highres.jpg" /> 图5.噬菌体ELISA筛选潜在ALS克隆（ALS与HT）。使用均质人体ALS，FTD和我们进行间接ELISA使用来自不同的克隆产生的噬菌体健康的脑组织（HT） 的样品。结果示<html>作为比对健康组织样本。结果表明，一些克隆TDP-43在ALS患者和那些在健康发现区分。在ALS，FTD和HT组织是从三个人的大脑样本（运动皮层）的组合。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52584/52584fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52584/52584fig6highres.jpg" /> 图6.潜在ALS克隆的噬菌体ELISA筛选（ALS与FTD）。在这里，我们将展示ALS的噬菌体ELISA结果与FTD的患者。大部分克隆的偏爱超过ALS FTD。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52584/52584fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html> _upload / 52584 / 52584fig7highres.jpg"/&gt; 潜在ALS克隆（ALS与HT）图7.单链抗体ELISA筛选，利用从每个克隆，我们可以观察到偏爱的TDP-43 ALS患者在健康的克隆产生的scFv的。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52584/52584fig7large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图8" src="/files/ftp_upload/52584/52584fig8highres.jpg" /> 潜在ALS克隆图8.单链抗体ELISA筛选（ALS与FTD）。我们的平移进程的成功比较ALS，以FTD在间接酶联免疫吸附不同的scFvs时进一步显现。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52584/52584fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。 。</a> 方法"&gt; <img alt="图9" src="/files/ftp_upload/52584/52584fig9highres.jpg" /> 图潜在ALS克隆人的9点印迹分析。使用点印迹，而不是ELISA是另一种技术，显示克隆的ALS在FTD或HT的特异性。克隆2A所示。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52584/52584fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Universidad San Sebastian

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

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer&#8217;s and Parkinson&#8217;s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

通过红外纳米光谱和原子力显微镜对单个蛋白质聚合进行特征化

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

科研

生物化学

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

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

矩阵辅助激光去离子成像质谱法显示人脑中的淀粉样β矿床

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

通过自由浮动免疫荧光测定,在皮外神经纤维中定位阿尔法核糖核酸聚合体

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%&#8211;20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

使用毛细血管电泳免疫测定来寻找人类血小板肌萎缩性侧索硬化的潜在生物标志物

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant ...

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

与神经退化有关的成人 果 蝇的可视化突触变性

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused ...

Nuclei Isolation and Super-Resolution Structured Illumination Microscopy for Examining Nucleoporin Alterations in Human Neurodegeneration

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). Collectively, these Nups function to regulate genome organization, gene expression, and nucleocytoplasmic transport (NCT). Recently, defects in NCT and alterations to specific Nups have been identified as early and prominent pathologies in multiple neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), Alzheimer&#39;s Disease (AD)/Frontotemporal Dementia (FTD), and Huntington&#39;s Disease (HD). Advances in both light and electron microscopy allow for a thorough examination of sub-cellular structures, including the NPC and its Nup constituents, with increased precision and resolution. Of the commonly used techniques, super-resolution structured illumination microscopy (SIM) affords the unparalleled opportunity to study the localization and expression of individual Nups using conventional antibody-based labeling strategies. Isolation of nuclei prior to SIM enables the visualization of individual Nup proteins within the NPC and nucleoplasm in fully and accurately reconstructed 3D space. This protocol describes a procedure for nuclei isolation and SIM to evaluate Nup expression and distribution in human iPSC-derived CNS cells and postmortem tissues.

This protocol describes an optimized workflow for nuclei isolation and super-resolution structured illumination microscopy to evaluate individual nucleoporins within the nucleoplasm and NPCs in induced pluripotent stem cell derived neurons and postmortem human tissues.

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

肌萎缩性侧索硬化症模型中突触功能的实时荧光测量

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae

Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between these features may be interrogated by controlling the phase transition of a disease-associated protein in a vulnerable cell type, although this experimental approach has been limited so far. Here, we describe a protocol to induce phase transition of the RNA/DNA-binding protein TDP-43 in spinal motor neurons of zebrafish larvae for modeling cytoplasmic aggregation of TDP-43 occurring in degenerating motor neurons in amyotrophic lateral sclerosis (ALS). We describe a bacterial artificial chromosome (BAC)-based genetic method to deliver an optogenetic TDP-43 variant selectively to spinal motor neurons of zebrafish. The high translucency of zebrafish larvae allows for the phase transition of the optogenetic TDP-43 in the spinal motor neurons by a simple external illumination using a light-emitting diode (LED) against unrestrained fish. We also present a basic workflow of live imaging of the zebrafish spinal motor neurons and image analysis with freely available Fiji/ImageJ software to characterize responses of the optogenetic TDP-43 to the light illumination. This protocol enables the characterization of TDP-43 phase transition and aggregate formation in an ALS-vulnerable cellular environment, which should facilitate an investigation of its cellular and behavioral consequences.

We describe a protocol to induce phase transition of TAR DNA-binding protein 43 (TDP-43) by light in the spinal motor neurons using zebrafish as a model.

TDP-43在斑马鱼幼虫脊髓运动神经元中的光遗传学相变

Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

来自大脑的淀粉样原纤维核心的生化纯化和蛋白质组学表征

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

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

摘要

探索更多视频

此视频中的章节

通过红外纳米光谱和原子力显微镜对单个蛋白质聚合进行特征化

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

矩阵辅助激光去离子成像质谱法显示人脑中的淀粉样β矿床

通过自由浮动免疫荧光测定,在皮外神经纤维中定位阿尔法核糖核酸聚合体

使用毛细血管电泳免疫测定来寻找人类血小板肌萎缩性侧索硬化的潜在生物标志物

与神经退化有关的成人果蝇的可视化突触变性

Nuclei Isolation and Super-Resolution Structured Illumination Microscopy for Examining Nucleoporin Alterations in Human Neurodegeneration

肌萎缩性侧索硬化症模型中突触功能的实时荧光测量

TDP-43在斑马鱼幼虫脊髓运动神经元中的光遗传学相变

来自大脑的淀粉样原纤维核心的生化纯化和蛋白质组学表征

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

摘要

探索更多视频

此视频中的章节

通过红外纳米光谱和原子力显微镜对单个蛋白质聚合进行特征化

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

矩阵辅助激光去离子成像质谱法显示人脑中的淀粉样β矿床

通过自由浮动免疫荧光测定,在皮外神经纤维中定位阿尔法核糖核酸聚合体

使用毛细血管电泳免疫测定来寻找人类血小板肌萎缩性侧索硬化的潜在生物标志物

与神经退化有关的成人 果 蝇的可视化突触变性

Nuclei Isolation and Super-Resolution Structured Illumination Microscopy for Examining Nucleoporin Alterations in Human Neurodegeneration

肌萎缩性侧索硬化症模型中突触功能的实时荧光测量

TDP-43在斑马鱼幼虫脊髓运动神经元中的光遗传学相变

来自大脑的淀粉样原纤维核心的生化纯化和蛋白质组学表征

与神经退化有关的成人果蝇的可视化突触变性