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

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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><tbody><tr><td>Chariot</td><td>Active Motif</td><td>30025</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Neurobasal medium</td><td>Life technologies</td><td>21103-049</td><td>warm up to 37 &amp;deg;C before using</td></tr><tr><td>1x B27</td><td>Life technologies</td><td>17504044</td><td>store at -20 &amp;deg;C</td></tr><tr><td>L-glutamine</td><td>Life technologies</td><td>25030-149</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Penicillin and Streptomycin</td><td>Life technologies</td><td>15140-122</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Imaris software</td><td>Bitplane</td><td>n.a.</td><td>expensive, but unmatched</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

Universidad de Cartagena UDEC

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

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

优化负染：通过电子显微镜检查小和不对称蛋白结构的高通量协议

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

科研

环境科学

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

细菌内膜显示筛查抗体片段的图书馆

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

生物化学

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

用奇米蛋白构建功能蛋白区的鉴定

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

监视分泌细菌毒力因子的组装使用站点特定的交联

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

免疫与感染

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The linker was created using molecular modeling software as a loop structure. Here, we introduce a protocol forin silico analysis of a complete scFv antibody that interacts with the epidermal growth factor receptor (EGFR). The homology modeling, with Pyrx of protein-protein docking and molecular dynamic simulation of the interacting scFv antibody and EGFR First, the authors used a protein structure modeling program and Python for homology modeling, and the antibody scFv structure was modeled for homology. The investigators downloaded Pyrx software as a platform in the docking study. The Molecular dynamic simulation was run using modeling software. Results show that when the MD simulation was subjected to energy minimization, the protein model had the lowest binding energy (-5.4 kcal/M). In addition, the MD simulation in this study showed that the docked EGFR-scFv antibody was stable for 20-75 ns when the movement of the structure increased sharply to 7.2 &#197;. In conclusion, in silicoanalysiswas performed, and the molecular docking and molecular dynamics simulations of the scFv antibody proved the effectiveness of the designed immune-therapeutic drug scFv as a specific drug therapy for EGFR.

<meta charset="utf8"></head><body>通过计算机模拟方法对表皮生长因子受体特异性单链片段变量进行结构分析

This antibody homology modeling prediction protocol is followed by antibody-receptor Pyrx docking and molecular dynamic simulation. These three primary methods are used to visualize the accurate antibody-receptor binding areas and the binding stability of the final structure.

通过 计算机 模拟方法对表皮生长因子受体特异性单链片段变量进行结构分析

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The ...

遗传学

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

生物学

通过抗体介导的实时成像增强蛋白质可视化

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

作者聚焦：揭示动物形态发生研究中机械和生化信号的动力学 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

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

摘要

探索更多视频

此视频中的章节

优化负染：通过电子显微镜检查小和不对称蛋白结构的高通量协议

细菌内膜显示筛查抗体片段的图书馆

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

用奇米蛋白构建功能蛋白区的鉴定

监视分泌细菌毒力因子的组装使用站点特定的交联

通过计算机模拟方法对表皮生长因子受体特异性单链片段变量进行结构分析

通过计算机模拟方法对表皮生长因子受体特异性单链片段变量进行结构分析

成像蛋白质 - 蛋白质相互作用在体内

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

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

摘要

探索更多视频

此视频中的章节

优化负染：通过电子显微镜检查小和不对称蛋白结构的高通量协议

细菌内膜显示筛查抗体片段的图书馆

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

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