Name: identification of functional protein regions through chimeric protein construction
Uploaded: 2019-01-08T15:00:00
Description: Watch this Scientific Journal Video about Identification of Functional Protein Regions Through Chimeric Protein Construction at JoVE.com

这项工作得到了马克斯·普朗克协会和舒赫特曼诊所 (德国巴特罗滕费尔德) 的支持。这项研究的一部分最初发表在《生物化学》杂志上。aderan-segarra, j. m., schindler, n., gajawada, p., lörchner, h., braun, t. & pöling, j。人组织受体激活需要人 oncostatin m (osm) 结合部位 iii 中的 ab 环路和 d-螺旋。j. biol. chem. 2018;18: 7017-7029. ©作者。

1. 奇美蛋白设计
<ol><li>选择合适的蛋白质 (供体) 与感兴趣的蛋白质 (接受者) 交换区域捐献者蛋白应该在结构上相似, 理想的情况下属于同一个蛋白质家族, 但缺乏生物活性作为读出。如果不知道与结构相关的蛋白质, 则可以使用自动工具 (如矢量对齐搜索工具 (vast)14、15)来识别潜在的候选元素<ol>
<li>访问蛋白质数据库 (pdb)16欧洲网站 (h...

<ol>
	<li>Huising, M. O., Kruiswijk, C. P., Flik, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+and+evolution+of+class-I+helical+cytokines.">Phylogeny and evolution of class-I helical cytokines.</a> The Journal of Endocrinology. 189 (1), 1-25 (2006).</li><li>Brocker, C., Thompson, D., Matsumoto, A., Nebert, D. W., Vasiliou, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+divergence+and+functions+of+the+human+interleukin+(IL)+gene+family.">Evolutionary divergence and functions of the human interleukin (IL) gene family.</a> Human Genomics. 5 (1), 30-55 (2010).</li><li>Bravo, J., Heath, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+recognition+by+gp130+cytokines.">Receptor recognition by gp130 cytokines.</a> The EMBO Journal. 19 (11), 2399-2411 (2000).</li><li>Schneider, G., Fechner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-based+de+novo.+design+of+drug-like+molecules.">Computer-based de novo. design of drug-like molecules.</a> Nature Reviews Drug Discovery. 4 (8), 649-663 (2005).</li><li>Heydenreich, F. M., Milju&#353;, T., Jaussi, R., Benoit, R., Mili&#263;, D., Veprintsev, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+mutagenesis+using+a+two-fragment+PCR+approach.">High-throughput mutagenesis using a two-fragment PCR approach.</a> Scientific Reports. 7 (1), 6787 (2017).</li><li>Adrian-Segarra, J. M., Schindler, N., Gajawada, P., L&#246;rchner, H., Braun, T., P&#246;ling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AB+loop+and+D-helix+in+binding+site+III+of+human+Oncostatin+M+(OSM)+are+required+for+OSM+receptor+activation.">The AB loop and D-helix in binding site III of human Oncostatin M (OSM) are required for OSM receptor activation.</a> The Journal of Biological Chemistry. 293 (18), 7017-7029 (2018).</li><li>Wang, Y., Pallen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+characterization+of+wild+type,+truncated,+and+mutant+forms+of+the+intracellular+region+of+the+receptor-like+protein+tyrosine+phosphatase+HPTP+beta.">Expression and characterization of wild type, truncated, and mutant forms of the intracellular region of the receptor-like protein tyrosine phosphatase HPTP beta.</a> The Journal of Biological Chemistry. 267 (23), 16696-16702 (1992).</li><li>Lim, J., Yao, S., Graf, M., Winkler, C., Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+analysis+of+full-length+midkine+reveals+novel+residues+important+for+heparin+binding+and+zebrafish+embryogenesis.">Structure-function analysis of full-length midkine reveals novel residues important for heparin binding and zebrafish embryogenesis.</a> The Biochemical Journal. 451 (3), 407-415 (2013).</li><li>Kim, K. -. W., Vallon-Eberhard, 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=In+vivo+structure/function+and+expression+analysis+of+the+CX3C+chemokine+fractalkine.">In vivo structure/function and expression analysis of the CX3C chemokine fractalkine.</a> Blood. 118 (22), 156-167 (2011).</li><li>Chollangi, S., Mather, T., Rodgers, K. K., Ash, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+loop+structure+in+oncostatin+M+determines+binding+affinity+toward+oncostatin+M+receptor+and+leukemia+inhibitory+factor+receptor.">A unique loop structure in oncostatin M determines binding affinity toward oncostatin M receptor and leukemia inhibitory factor receptor.</a> The Journal of Biological Chemistry. 287 (39), 32848-32859 (2012).</li><li>Aasland, D., Schuster, B., Gr&#246;tzinger, J., Rose-John, S., Kallen, K. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+leukemia+inhibitory+factor+receptor+functional+domains+by+chimeric+receptors+and+cytokines.">Analysis of the leukemia inhibitory factor receptor functional domains by chimeric receptors and cytokines.</a> Biochemistry. 42 (18), 5244-5252 (2003).</li><li>Hermanns, H. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NCBI's+LocusLink+and+RefSeq.">NCBI's LocusLink and RefSeq.</a> Nucleic Acids Research. 28 (1), 126-128 (2000).</li><li>Sievers, F., Wilm, 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=Fast,+scalable+generation+of+high-quality+protein+multiple+sequence+alignments+using+Clustal+Omega.">Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.</a> Molecular Systems Biology. 7, 539 (2011).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108 (2), 193-199 (1991).</li><li>Barbas, C. 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嵌合蛋白的产生构成了一种多功能的技术, 它能够超越截断蛋白的限制, 以解决细胞因子受体结合域的模块化等问题13。嵌合体的设计是这种研究的关键一步, 需要仔细考虑。建立功能域的初步研究一般需要在第一阶段替换广泛的区域, 而较小的可变长度的替换则更适合于对单一区域的详细研究。在这一步中, 应特别注意蛋白质家族中存在的小的保守图案, 因为这些图案往往表明功?...

几种类型的蛋白质, 包括细胞因子和生长因子, 被分组在家庭的成员有相似的三维结构, 但往往发挥不同的生物功能1,2。这种功能多样性通常是分子活性位点3中氨基酸组成差异小的结果.确定这些位点和功能决定因素不仅提供了宝贵的进化见解, 而且还设计了更具体的激动剂和抑制剂4。然而, 在与结构相关的蛋白质之间经常发现的残留物成分的大量差异使这项任务变得复杂。尽管构建包含数百个突变体的大型库现在是可行的, 但评估每一个残留物的变化和组合仍然是一项具有挑战性和耗时的工作5。
因此, 评估大型蛋白质区域的功能重要性的技术对于将可能的残留物数量减少到可管理的第6种是有价值的.截断蛋白一直是解决这一问题最常用的方法。因此, 如果所研究的蛋白质功能受到特定区域7、8、9的删除的影响, 则区域在功能上被认为是相关的。然而, 这种方法的一个主要局限性是, 缺失会影响蛋白质的二级结构, 导致错误折叠、聚集和无法研究预期的区域。一个很好的例子是细胞因子在成本蛋白 m (osm) 上的截断版本, 其中一个内部删除大于7个残留物导致一个错误折叠的突变体, 不能进一步研究10。
嵌合蛋白的产生是一种替代和创新的方法, 允许分析更大的蛋白质区域。这种方法的目的是通过另一种蛋白质中与结构相关的序列交换对蛋白质感兴趣的区域, 以评估被替换的部分对特定生物功能的贡献。在信号受体领域广泛用于识别功能域11,12, 嵌合蛋白是特别有用的研究蛋白质家族的氨基酸认同少, 但保守的二级结构。在白细胞介素-6 (il-6) 类细胞因子类中可以找到适当的例子, 如白细胞介素-6 和睫毛神经营养因子 (6% 序列识别)13或白血病抑制因子 (lif) 和 osm (20% 身份)6, 其中下面的协议是基于。

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Labcycler thermocycler</td>
			<td style="width:97px;">Sensoquest</td>
			<td style="width:119px;">011-103</td>
			<td style="width:167px;">Any conventional PCR machine can be employed to carry out this protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NanoDrop 2000c UV-Vis spectrophotometer</td>
			<td style="width:97px;">ThermoFisher Scientific</td>
			<td style="width:119px;">ND-2000C&#160;</td>
			<td>DNA quantification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GeneRuler 100 bp DNA ladder</td>
			<td style="width:97px;">ThermoFisher Scientific</td>
			<td style="width:119px;">SM0241</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GeneRuler DNA Ladder Mix</td>
			<td style="width:97px;">ThermoFisher Scientific</td>
			<td style="width:119px;">SM0331</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AscI restriction enzyme</td>
			<td style="width:97px;">New England Biolabs</td>
			<td style="width:119px;">R0558</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PacI restriction enzyme</td>
			<td style="width:97px;">New England Biolabs</td>
			<td style="width:119px;">R0547</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phusion Hot Start II DNA Polymerase</td>
			<td style="width:97px;">ThermoFisher Scientific</td>
			<td style="width:119px;">F-549S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTP set (100 mM)</td>
			<td style="width:97px;">Invitrogen</td>
			<td>10297018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 DNA ligase</td>
			<td style="width:97px;">Promega</td>
			<td style="width:119px;">M1804</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NucleoSpin Gel and PCR clean-up kit</td>
			<td style="width:97px;">Macherey-Nagel</td>
			<td align="right" style="width:119px;">740609</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">MGC Human LIF Sequence-Verified cDNA (CloneId:7939578), glycerol stock</td>
			<td style="width:97px;">ThermoFisher Scientific</td>
			<td style="width:119px;">MHS6278-202857165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LE agarose</td>
			<td style="width:97px;">Biozym</td>
			<td align="right" style="width:119px;">840004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primers</td>
			<td style="width:97px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td>Custom order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Human Oncostatin M cDNA</td>
			<td style="width:97px;"></td>
			<td style="width:119px;"></td>
			<td>Gift of Dr. Heike Hermanns (Division of Hepatology, University Hospital W&#252;rzburg, Germany)&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">pCAGGS vector with PacI and AscI restriction sites</td>
			<td style="width:97px;"></td>
			<td style="width:119px;"></td>
			<td>Gift of Dr. Andr&#233; Schneider (Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany)</td>
		</tr>
	</tbody>
</table>

identification of functional protein regions through chimeric protein construction

该协议的目标包括设计嵌合蛋白, 在这种蛋白质的不同区域被结构相似的蛋白质中的相应序列所取代, 以确定这些区域的功能重要性。这种嵌合体是通过嵌套 pcr 协议产生的, 该协议使用重叠的 dna 片段和经过充分设计的引物, 然后在哺乳动物系统中表达, 以确保本地二级结构和翻译后的修饰。
然后, 在适当的读出分析中, 嵌合体的活性丧失表明了不同区域的功能作用。因此, 确定了隐藏一组关键氨基酸的区域, 这些区域可以通过互补技术 (例如位向诱变) 进一步筛选, 以提高分子分辨率。虽然仅限于可以发现功能不同的结构相关蛋白质的情况, 但嵌合蛋白已被成功地用于识别蛋白质 (如细胞因子和细胞因子受体) 中的关键结合区域。这种方法特别适用于蛋白质的功能区域没有得到很好界定的情况, 是定向进化方法的宝贵的第一步, 目的是缩小感兴趣的区域, 减少所涉及的筛选工作。

嵌合蛋白的构建和生成 (图 1) 将以白细胞介素-6 细胞因子家族的两名成员 osm 和 lif 为例,这是最近发表的一项研究的主题6。图 2显示了这些蛋白质的三维结构。这两个分子都采用 i 类细胞因子的特征二级结构, 四个螺旋 (称为 a 到 d) 被捆绑起来, 并由循环 28连接。人类蛋白质的对齐氨基酸结构?...

Watch this Scientific Journal Video about Identification of Functional Protein Regions Through Chimeric Protein Construction at JoVE.com

Identification of Functional Protein Regions Through Chimeric Protein Construction

结构上相关的蛋白质经常发挥不同的生物学功能。交换这些蛋白质的等效区域, 以创造嵌合蛋白是一种创新的方法, 以确定关键的蛋白质区域, 负责其功能分化。

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 ...

交换结构上相似的蛋白质区域来产生基梅拉，使我们能够研究负责分子生物功能的区域的重要性。区域交换是蛋白质的一般二级结构，因此该区域对蛋白质整体结构的重要结构可以与直接调解生物功能的二级结构区分开来。它可以转换为选择要替换的确切区域。从较大的交换开始通常有助于识别蛋白质的关键功能。要开始该程序，请选择合适的蛋白质作为捐赠者，以交换与文本协议中详述的感兴趣的蛋白质的区域。然后，首先访问 ref seq网页中的基因部分，从参考序列数据库中获取接受者和供体蛋白质的蛋白质氨基酸序列。在搜索框中键入感兴趣的蛋白质的名称，然后单击搜索。单击结果列表中所需物种的基因名称。向下滚动到 ref seq 部分以查看所有记录的等值形式。单击感兴趣的等式序列标识符。向下滚动并单击 CD 以突出显示基因的蛋白质编码区域。在屏幕的右下角，单击 FAFSDA 并复制基因序列。使用合适的DNA编辑软件保存DNA序列。使用免费可用的 APE 时，打开程序，将复制的序列粘贴到空白框中，选择序列名称，然后单击"保存"。现在，选择在不同的基美利构造中要替代的蛋白质区域，首先将感兴趣的蛋白质序列划分在不同的结构区域。为此，请从PDBe网站下载感兴趣的蛋白质的结构数据。访问蛋白质的 PDBe 页面，通过单击屏幕右侧的下载下载来下载 PDBe 文件。在分子可视化系统中打开 PDBe 文件，如 Pymol。在Pymol中，显示核苷酸序列，隐藏默认结构数据并选择卡通视图，以清楚地显示蛋白质的结构特征。单击屏幕顶部的核苷酸序列以突出显示分子的不同部分，注意与每种独特结构特征对应的氨基酸。现在，通过打开DNA序列，选择它，点击 ORF 翻译，对 APE 中 DNA 序列中不同的结构区域进行注释，然后单击第一个区域的最后一个氨基酸，以突出显示其在 DNA 序列中的位置，并选择该区域的核苷酸编码。右键单击所选内容并选择新功能，为其命名和颜色。对上一步中标识的每个结构区域重复此过程。为了对齐两种蛋白质的残余序列，首先，获取APE中供体和受体蛋白的完整氨基酸序列。和以前一样，打开捐赠者 DNA 序列，选择它，然后单击 ORF 翻译。然后，访问Clustal欧米茄网页，然后把两种蛋白质的氨基酸序列。每个序列应按一条文本行进行，该文本行应正确识别蛋白质名称。向下滚动并单击"提交"。通过单击下载对齐文件选项卡检索对齐文件并保存它。此文件可由任何文本编辑程序打开。使用对齐文件作为参考，注释其DNA序列中供体蛋白质的相应结构区域。创建受体蛋白的带注释DNA序列的副本，并将其重命名为基美蛋白。在 APE 中打开重命名的 DNA 序列。选择在供体蛋白中交换的区域，然后在重命名的受体蛋白中选择相应的区域。粘贴供体蛋白DNA序列，然后保存变化。使用 DNA 编辑器（如 APE）设计终端底像。创建新的 DNA 文件，使用前线序列启动端端底转。其次是在向量MCS中选择的第一个限制位点和在感兴趣的基因的初始18至27个碱基对中选择的可选分空间。在新的DNA文件中，设计C终端底转序列，从感兴趣的基因的最终18到27个碱基对开始，然后是可选的分位空间、选择的第二个限制位点和领导序列。突出显示整个序列，右键单击，并选择反向恭维以获取反向底图。现在，为基梅里奇构造中每个边界区域设计底木。在 kimera 的 DNA 序列中，突出显示原始序列和插入序列接触的区域中的 30 个碱基对区域。这由每个序列中的 15 个基对组成。复制该区域，并将其粘贴到新的 DNA 文件中。此序列将是前进底图。复制上一步生成的前进底像，并将其重命名为反向底项。突出显示底转序列，右键单击，并选择反向恭维以生成反向底转序列。对基美脱氧核酸序列中的每个接触区重复这些步骤。通常，需要两组正向反向底转才能生成一个 kimera，除非更换发生在 N 端子或 C 终端区域。为组成基美蛋白的每个片段准备单独的PCR反应混合物。首先在冰上设置一点五毫升微注入管，然后按文本协议中列出的顺序对PCR混合物的不同试剂进行移液。确保每个 PCR 反应都使用正确的底向和模板。标记两个薄壁零点两个毫升PCR管每个反应，并转移20微升的相应PCR混合物在每个管。将 PCR 管传输到 PCR 热循环器中，并启动文本协议中详细说明的协议。PCR反应完成后，在每个管中加入四个微升的六个xDNA加载缓冲液。将 1% 的 agaross 凝胶插入电泳单元中，并盖上 TAE 缓冲液。小心地将样品与分子量一起装入凝胶中。在以 80 至 120 伏运行凝胶 20 至 45 分钟后，关闭电泳单元并取出前毛凝胶。在紫外光下可视化放大的DNA波段。使用剃须刀刀片，从凝胶中切出单个DNA片段，并转移到标有两个民兵微注入管。使用 PCR 清理套件净化 DNA 片段后，通过测量光谱仪中 260 纳米和 340 纳米样品的吸光度来量化提取的 DNA 量。现在，执行PCR扩增以生成基美脱DNA序列。首先，设置50微升的PCR反应，以熔合基梅拉的单独成分一样， 像以前一样。使用 N 端子和 C 端子底像以及每个放大 DNA 片段的 10 纳米图。恢复和量化纯化DNA片段一样，在30微升的原子核自由水。此片段包含由终端底像中包含的限制位点所侧侧的 kimeric DNA 序列。一代基美尔蛋白的例子是两个成员之间的卢克和六个西托金家族。白血病抑制因子中的上质M。OSM 提升 kimera 是 OSM 的 BC 环路区域与相应的提升序列交换的结果。第一个PCR扩增步骤由三个独立的反应组成。N 终端 OSM 片段，需要 N 终端 OSM 转发和 BC 启动反向源使用 OSM 作为模板。利用 LIF 作为模板，通过 BC 开始向前和 BC N 反向底转获得 LIF BC 环路。C 终端 OSM 片段使用 BC 端前和 C 终端 OSM 反向底转以及 OSM 作为模板。然后，这些纯化片段在第二次PCR反应中用作模板，同时使用N-终端OSM前进和C-终端OSM反向源器，以放大相应的OSM LIF BC环路基因序列。在纯化和转化为电子大肠杆菌后，通过限制性酶消化分离和筛选单个质粒，以正确插入DNA片段。凝胶电泳显示阳性命中，然后发送序列验证。基默尔蛋白质必须产生，活动在一个完美的派系系统中测量。为了确定被替换区域对该特定派别的重要性。该技术已非常有用，并经常应用，干扰信号受体，以识别关键功能域和受体识别位点。我用于DNA电泳的号码应该使用一次性手套、实验室外套和防护眼镜处理。

Research

JoVE Journal

Biochemistry

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

科研

生物化学

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

化细胞穿透肽研究胞内蛋白-蛋白质相互作用

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Evaluation of ProteinÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Protein Interactions using an On-Membrane Digestion Technique

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...