所提出的工作是由健康研究加拿大学院资助的部分支持/加拿大合作计划，RD的脆性X研究基金会，杰罗姆勒琼基金会RD时，InterdisziplinäresZentrum酒店献给大学埃尔兰根 - 纽伦堡的klinische Forschung到RD和从德意志研究联合会为RD和RE。作者要感谢英格里德曾格与细胞培养技术维修援助以及用于制作pCMV5-FLAG载体可M. Wegner的教授。作者进一步想特别感谢娜佳施罗德在视频拍摄方面有帮助的支持。

1.股票方案<ol><li>重悬在无菌小时冻干活性基序粉末2 O至2微克/微升的最终浓度。仔细挖掘混合。 </li><li>制备小等分试样（ 例如，每12微升，2微升被每个反应需要）并将其存储在-20℃。 </li></ol> 2.细胞的制备<ol><li>种子哺乳动物细胞如24孔板于500μl生长培养基包括抗生素对HEK293细胞或原代神经元。 注意:当使用神经元，种子细胞以低密度并且仅使用24孔板的两个中间行。因此，每口井将有一个空的对应孵化（步骤4.2）时怀有网上平台。当处理的神经元，始终确保细胞没有保持孵化器外超过几分钟。 </li><li>培养的细胞在适当的条件（加湿5％CO 2和37℃），直到细胞是应用程式。 50％汇合。 注意:如果使用的神经元， 培养细胞，直到所需的发育阶段达到并改变应用程序。介质的25-50％，每周两次。介质:Neurobasal培养基（含有1x B27，5mM的L-谷氨酰胺和1x青霉素和链霉素;与参考16进行比较）。 </li></ol> 3.战车复杂地层<ol><li>每个反应，稀释分别POI的或相应的抗体，以及作为对照蛋白或对照抗体的0.1-2微克，在50μlPBS中。 注:该技术也适用于由预先绑定第一和第二抗体（CP&#39;代表结果"， 图1）的大分子复合物。混合和旋转。 </li><li>对于每个样品，以避免战车的自缔合使用单独的试管稀释在50μlPBS中的2微升战车原液。混合和旋转。 </li><li>用移液管转移稀释的POI或抗体（步骤3.1）的战车稀释婷。混合和旋转。 </li><li>孵育在室温下该混合物30分钟（战车-POI络合物将形成）。 </li></ol> 4.转染细胞<ol><li>稀释战车配合于100μl预热的生长培养基中（37℃，无添加剂）（步骤3.4）。除去培养基并洗一次使用预热的1×PBS中的细胞。 注:当使用神经元，不丢弃介质，它会被重复使用。保持在37℃。对于洗涤，使用含有0.5毫米氯化镁2和氯化钙 PBS。 建议:保持中等井空，同时培养板（步骤4.5）。 </li><li>应用战车络合物溶液（步骤4.1）至细胞中并轻轻摇动细胞以确保均匀的溶液分布。生长的标准条件（步骤2.2）下的细胞1小时。添加血清的10％的最终浓度。当使用神经元， 从步骤4.1中添加介质。生长细胞为1至2小时。 注意:最佳孵育时间取决于大小和货物的性质，可能需要根据经验调整。以下建议可以作为一个准则:对于肽，1小时通常是足够的，对于蛋白质被推荐1-2小时，对抗体2小时，并为神经元的转染抗体4小时。 </li><li>流程细胞分析如常。请注意:该技术是用固定的协议以及现场成像实验兼容。 </li></ol> 5.成像<ol><li>使用激光扫描显微镜，使用大约0.25-0.5微米的距离取细胞和/或感兴趣的细胞区室的Z轴堆叠。精确的层距离取决于要重建所述结构的尺寸和需要被单独地确定。 </li></ol> 6.重建<ol><li>在下面的步骤，使用Esc键底部选择和导航，CNTR之间切换通过单击以选中多个对象O.ñ他们和Shift键削减对象（CP。步骤6.10）。 </li><li>打开了Imaris的LSM-文件。 </li><li>使用显示器调整为每个通道，对比照片，直到最亮的结构达到饱和。请注意，本次调整不会影响面施工，它只会帮助实验者在阈值图像。 </li><li>点击添加新曲面图标。一个向导将打开。 </li><li>选择:段唯一的兴趣的一个区域。继续进行下一步骤。 </li><li>调整选择框的边缘，以适应您感兴趣的对象。请记住图像具有3个维度，而且选择必须在z平面和调整。如果选择框的矩形形状应该没有正确的感兴趣的对象相匹配，将对象和/或放大的方块，直至所有所需的结构被嵌入。不需要的对象可能会稍后删除（CP。步骤6.10）。继续。 </li><li>选择合适的通道annel。外围应用详细程度或球体直径应设置为与正在重建结构。根据信号的特性，可以使用任一绝对强度或局部对比度。一般情况下，局部对比度适用于扩散信号更好。在任何情况下，该设置需要分别为每个感兴趣的信号或结构，分别进行调整。 </li><li>使用阈值工具，重建的感兴趣结构。 </li><li>通过点击绿色箭头底完成重建。 </li><li>通过使用铅笔工具切割并根据需要去除表面进一步调整的对象。它是唯一可能在南北方向上切断，因此，倾斜图像​​，以切割所需的对象。 </li><li>通过选择统计，详细统计数据和平均值获得每个表面体积，面积测量和强度。 </li><li> （可选步骤）观察颜色编码的统计数据（CP。 图2E </s仲&gt;和F），选择相应的表面的颜色标签和标记"统计编码&#39;而不是&#39;基地&#39;。几个选项将被显示。 </li></ol>

<ol>
	<li>Santoro, M. R., Bray, S. M., Warren, S. T., Abbas, A. K., Galli, S. J., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Annual Review of Pathology: Mechanisms of Disease. 7, 219-245 (2012).</li><li>Jiang, T. F., Chen, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfunction+of+two+lysosome+degradation+pathways+of+alpha-synuclein+in+Parkinson's+disease:+potential+therapeutic+targets?.">Dysfunction of two lysosome degradation pathways of alpha-synuclein in Parkinson's disease: potential therapeutic targets?.</a> Neurosci Bull. 28, 649-657 (2012).</li><li>Howlett, D. R., Richardson, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+of+APP+transgenic+mice:+a+model+of+Alzheimer's+disease+or+simply+overexpression+of+APP?.">The pathology of APP transgenic mice: a model of Alzheimer's disease or simply overexpression of APP?.</a> Histol Histopathol. 24, 83-100 (2009).</li><li>Carlson, G. 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=Genetic+Modification+of+the+Phenotypes+Produced+by+Amyloid+Precursor+Protein+Overexpression+in+Transgenic+Mice.">Genetic Modification of the Phenotypes Produced by Amyloid Precursor Protein Overexpression in Transgenic Mice.</a> Human molecular genetics. 6, 1951-1959 (1997).</li><li>Jiao, X. F., Wang, Z., Kiledjian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+mRNA-secapping+regulator+implicated+in+X-linked+mental+retardation.">Identification of an mRNA-secapping regulator implicated in X-linked mental retardation.</a> Mol. Cell. 24, 713-722 (2006).</li><li>Fukami, 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+member+of+a+gene+family+on+Xp22.3,+VCX-A,+is+deleted+in+patients+with+X-linked+nonspecific+mental+retardation.">A member of a gene family on Xp22.3, VCX-A, is deleted in patients with X-linked nonspecific mental retardation.</a> Am. J. Med. Genet. 67, 563-573 (2000).</li><li>Van Esch, 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=Deletion+of+VCX-A+due+to+NAHR+plays+a+major+role+in+the+occurrence+of+mental+retardation+in+patients+with+X-linked+ichthyosis.">Deletion of VCX-A due to NAHR plays a major role in the occurrence of mental retardation in patients with X-linked ichthyosis.</a> Human molecular genetics. 14, 1795-1803 (2005).</li><li>Sebat, 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=Strong+association+of+de+novo+copy+number+mutations+with+autism.">Strong association of de novo copy number mutations with autism.</a> Science. 316, 445-449 (2007).</li><li>Tartaglia, G. G., Pechmann, S., Dobson, C. M., Vendruscolo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+on+the+edge:+a+link+between+gene+expression+levels+and+aggregation+rates+of+human+proteins.">Life on the edge: a link between gene expression levels and aggregation rates of human proteins.</a> Trends Biochem Sci. 32, 204-206 (2007).</li><li>Schwanhausser, B., 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=Global+quantification+of+mammalian+gene+expression+control.">Global quantification of mammalian gene expression control.</a> Nature. 473, 337-342 (2011).</li><li>Uchino, K., Ochiya, T., Takeshita, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+therapeutics+and+applications+of+microRNAs+in+cancer+treatment.">RNAi therapeutics and applications of microRNAs in cancer treatment.</a> Jpn J Clin Oncol. 43, 596-607 (2013).</li><li>Li, T., Wu, M., Zhu, Y. Y., Chen, J., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+RNA+interference-based+therapeutics+and+application+of+multi-target+small+interfering+RNAs.">Development of RNA interference-based therapeutics and application of multi-target small interfering RNAs.</a> Nucleic Acid Ther. 24, 302-312 (2014).</li><li>DeVincenzo, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise,+pitfalls+and+progress+of+RNA-interference-based+antiviral+therapy+for+respiratory+viruses.">The promise, pitfalls and progress of RNA-interference-based antiviral therapy for respiratory viruses.</a> Antivir Ther. 17, 213-225 (2012).</li><li>Fellmann, C., Lowe, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+RNA+interference+rules+for+silencing.">Stable RNA interference rules for silencing.</a> Nat Cell Biol. 16, 10-18 (2014).</li><li>Morris, M. C., Depollier, J., Mery, J., Heitz, F., Divita, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peptide+carrier+for+the+delivery+of+biologically+active+proteins+into+mammalian+cells.">A peptide carrier for the delivery of biologically active proteins into mammalian cells.</a> Nat Biotechnol. 19, 1173-1176 (2001).</li><li>Derlig, K., Gie&#223;l, A., Brandst&#228;tter, J. H., Enz, R., Dahlhaus, R. <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+Characterisation+of+Simiate,+a+Novel+Protein+Linked+to+the+Fragile+X+Syndrome.">Identification and Characterisation of Simiate, a Novel Protein Linked to the Fragile X Syndrome.</a> PLoS One. 8, e83007 (2013).</li><li>Derlig, K., 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=Simiate+is+an+Actin+binding+protein+involved+in+filopodia+dynamics+and+arborisation+of+neurons.">Simiate is an Actin binding protein involved in filopodia dynamics and arborisation of neurons.</a> Frontiers in Cellular Neuroscience. 8, (2014).</li><li>Loudet, 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=Non-covalent+delivery+of+proteins+into+mammalian+cells.">Non-covalent delivery of proteins into mammalian cells.</a> Org Biomol Chem. 6, 4516-4522 (2008).</li><li>Nagel, 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=Polymeric+microspheres+as+protein+transduction+reagents.">Polymeric microspheres as protein transduction reagents.</a> Mol Cell Proteomics. 13, 1543-1551 (2014).</li><li>Selim, A. 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=Anti-osteoactivin+antibody+inhibits+osteoblast+differentiation+and+function+in+vitro.">Anti-osteoactivin antibody inhibits osteoblast differentiation and function in vitro.</a> Crit Rev Eukaryot Gene Expr. 13, 265-275 (2003).</li><li>Jain, A., Brady-Kalnay, S. M., Bellamkonda, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Rho+GTPase+activity+alleviates+chondroitin+sulfate+proteoglycan-dependent+inhibition+of+neurite+extension.">Modulation of Rho GTPase activity alleviates chondroitin sulfate proteoglycan-dependent inhibition of neurite extension.</a> J Neurosci Res. 77, 299-307 (2004).</li><li>Watanabe, S., Wakasugi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+function+of+human+neuroglobin+is+correlated+with+its+guanine+nucleotide+dissociation+inhibitor+activity.">Neuroprotective function of human neuroglobin is correlated with its guanine nucleotide dissociation inhibitor activity.</a> Biochem Biophys Res Commun. 369, 695-700 (2008).</li><li>Eguchi, 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=Optimization+of+nuclear+localization+signal+for+nuclear+transport+of+DNA-encapsulating+particles.">Optimization of nuclear localization signal for nuclear transport of DNA-encapsulating particles.</a> J Control Release. 104, 507-519 (2005).</li></ol>

The authors have nothing to disclose.

在这里，我们提出了一个协议，以研究在控制剂量驱动的方式细胞功能的蛋白表达水平的意义。描述该协议允许在各种哺乳动物细胞类型，包括海马神经元蛋白表达的微调操作，从而促进细胞水平上的蛋白质功能的详细研究。 虽然RNA干扰表示公知的策略来下调的POI，它有它的缺点（CP 14）。不仅是高度特异性的和有效的序列的识别可能是昂贵和费时的，而且还因为毒?...

蛋白表达的严格控制是必不可少的每一个生物体指挥自身的发展，以及对环境的信号作出反应。因此，各种机制的许多已在进化过程中发明了精确地调节由该应用编码各蛋白的表达水平。 20000个基因在其生命中的任何给定时间存在于任何真核细胞。发生在蛋白质生产的不同阶段，调节机制的范围从染色质结构，转录和RNA的管理处理，以的翻译后蛋白质修饰，转运和降解的方向。 因此，并不奇怪，在潜在的分子机械和改变蛋白质表达水平的故障已经与不同的疾病，如癌症或智障相关联。的确，看着神经元的发育和哺乳动物大脑功能的优秀的复杂性，森西这些复杂的系统的拉了一改建中蛋白表达表现在以下几个知名知识分子的赤字包括老年痴呆和帕金森病（AD和PD），以及自闭症谱系障碍（ASD）之类的脆性X综合征（FXS）。后者疾病的特征在于多种蛋白，这是由于单个翻译调节蛋白，FMRP（脆性X智力低下蛋白）1-4的损失广泛错误表达。此外，影响变电荷染色体重排X连锁蛋白A（VCXA），蛋白质，它管理通过修饰mRNA的加帽5 mRNA的稳定性和翻译，最近已经智力缺陷有关，而点突变未在患者确定有认知障碍截至目前6，7，这表明所观察到的精神损伤从改变VCXA表达及其靶prote的失调表达起源插件。在与这些发现，研究调查是否从头与ASD有关的基因的拷贝数变异建立的新基因重复和缺失是ASD 8显著危险因素，从而支持的想法，升高的或降低的蛋白质表达水平可能引起线智力缺陷。 值得注意的是，最近的研究还提供了证据，给定蛋白质的表达水平被精确地调节，以防止其作为高蛋白量的结果聚合几乎没有安全余量​​9。因此，已经提出，即使是很小的增量是足以诱导疾病如AD和PD 9。虽然各种促进蛋白质表达控制分子机器的显示在这些发现的光线复杂的调节方案，一项研究调查了5000哺乳动物基因10的表达水平证明本质上优选更简约的方案:蛋白质的细胞大量被证明在翻译10的水平主要是调节，从而说明该RNA的可用性管理，主要用来微调蛋白的表达。 研究的目的蛋白质的剂量（POI的），因此不仅是组成的蛋白质的内源功能的理解重要的，但也对许多疾病的调查和疗法的发展。因此，过去的十年里，利用RNA干扰来操纵POI用量几种策略的推进。虽然RNA干扰被广泛用于研究蛋白质的功能，并且即使在临床试验中被用于治疗癌症或眼疾病以及追求患者11-13抗病毒治疗，一些困难可能出现可能使战略是不可能的。例如，该种子序列，其中通过同源驱动击倒是对比试验BLE短，因此促进脱靶效应。由于高效的序列是罕见的，需要在数千的选项（在14中综述）被发现，确定适当的序列可以是费时和昂贵的，但结果仍可能会令人失望。 另一种策略是直接针对被抗体的POI。这里，我们说明了使用蛋白质载体战车（由Active Motif的制造）以降低细胞蛋白的可用性，和三维重建的就业研究蛋白质功能下列击倒。 战车，本身2.8 kDa的肽的活性基序，是用来梭肽，蛋白质和抗体穿过哺乳动物细胞15的细胞膜。与POI的肽相关联通过形成利用疏水相互作用，由此战车-POI络合物非共价地偶联的大分子复合物内化到细胞中内体-INDependent方式。重要的是，战车被指示为既不影响穿梭蛋白的细胞内定位，也发挥细胞毒性作用或影响其货物15的生物学活性。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chariot</td>
			<td>Active Motif</td>
			<td>30025</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life technologies</td>
			<td>21103-049</td>
			<td>warm up to 37&#176;C before using</td>
		</tr>
		<tr>
			<td>1xB27</td>
			<td>Life technologies</td>
			<td>17504044</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Life technologies</td>
			<td>25030-149</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>&#160;Penicilline and Streptomycine</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td>n.a.</td>
			<td>expensive, but unmatched</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>n.a.</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying protein function and the role of altered protein expression by antibody interference and three-dimensional reconstructions

表达了严格的管理不仅是必不可少的每一个活着的有机体，而且要调查细胞模型蛋白质功能的重要策略。因此，最近的研究发明了不同的工具来靶向哺乳动物细胞系，甚至动物模型，包括RNA和抗体干扰蛋白的表达。虽然第一战略在过去的二十年中收集的关注，肽介导穿过细胞膜进入细胞抗体货物的转运，得到少得多的利息。在此出版物中，我们提供了一个详细的协议如何利用在人胚肾细胞，以及在原代海马神经元命名战车的肽载体进行抗体干扰实验和进一步说明在分析蛋白质功能的三维重建的应用。我们的研究结果表明，战车是，可能是由于它的核定位信号，particulaRLY非常适合于目标驻留在体细胞和细胞核蛋白。值得注意的是，将战车初级海马培养时，试剂竟然是出奇地好，通过分离的神经元接受。

在下面的段落，显示了POI的功能击倒示范结果使用战车试剂和抗体的干扰呈现（Simiate，进一步的详细信息，请参阅16，17）。的研究结果表明，Simiate的减弱表达损害转录活性，而且，在剂量依赖的方式，诱导细胞凋亡，在超过99％的死亡率达到高潮，如果施加更高的抗体量（&gt; 1微克抗体）（这些发现以前出版16）。在这里，我们现在提出详细使用的技术和协议，?...

Watch this Scientific Journal Video about Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions at JoVE.com

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

研究蛋白质功能和改变的蛋白的表达由源抗体干扰和三维重建中的作用

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

studying-protein-function-role-altered-protein-expression-antibody

Research

JoVE Journal

Bioengineering

6.6K 次观看.  University of Erlangen-Nuremberg. Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

视频: Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

荧光光谱法测定膜蛋白质折叠的热力学

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

科研

生物工程

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

研究蛋白质折叠的微流体混合器

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

研究模型声乐折叠聚合物引起的三维流量分离

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

烟草中瞬时表达的蛋白作为一个案例研究:复杂系统采用的实验方法设计的表征

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.