Name: studying protein import into chloroplasts using protoplasts
Uploaded: 2018-12-10T13:00:00
Description: Watch this Scientific Journal Video about Studying Protein Import into Chloroplasts Using Protoplasts at JoVE.com

这项工作是在农业科技发展合作研究方案 (项目号) 的支持下进行的。pj010953012018), 政府农村发展管理局和国家研究基金会 (韩国) 赠款, 由科学和信息和通信技术部资助 (no. 2016r1e1a1a02922014), 大韩民国。

1. 拟南芥植物的生长
<ol><li>制备1升 gamborg b5 (b5) 培养基, 加入3.2 克 b5 培养基, 包括维生素, 20 克蔗糖, 0.5 克 2-(n-吗啡) 乙烷磺酸 (mes) 约800毫升去离子水, 并调整 ph 值到5.7 与氢氧化钾 (koh)。加入更多的去离子水, 使总体积在121°c 下达到1升, 加入8克植物琼脂和高压灭菌器15分钟。</li>
	<li>允许介质冷却至 55°c, 并将 b5 介质的 20–25 ml 倒入培养皿中 (直径9厘米, 高度1.5 厘米) 在干净的长凳上。干板 2-3天...

<ol>
	<li>Jarvis, P., Lopez-Juez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+homeostasis+of+chloroplasts+and+other+plastids.">Biogenesis and homeostasis of chloroplasts and other plastids.</a> Nature Reviews Molecular Cell Biology. 14 (12), 787-802 (2013).</li><li>Neuhaus, H. E., Emes, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonphotosynthetic+Metabolism+in+Plastids.">Nonphotosynthetic Metabolism in Plastids.</a> Annual Review of Plant Physiology and Plant Molecular Biology. 51, 111-140 (2000).</li><li>Rolland, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biosynthetic+capacities+of+the+plastids+and+integration+between+cytoplasmic+and+chloroplast+processes.">The biosynthetic capacities of the plastids and integration between cytoplasmic and chloroplast processes.</a> Annual Review of Genetics. 46, 233-264 (2012).</li><li>Jarvis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+nucleus-encoded+proteins+to+chloroplasts+in+plants.">Targeting of nucleus-encoded proteins to chloroplasts in plants.</a> New Phytologist. 179 (2), 257-285 (2008).</li><li>Li, H. M., Chiu, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Transport+into+Chloroplasts.">Protein Transport into Chloroplasts.</a> Annual Review of Plant Biology. 61, 157-180 (2010).</li><li>Keegstra, K., Cline, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+import+and+routing+systems+of+chloroplasts.">Protein import and routing systems of chloroplasts.</a> Plant Cell. 11 (4), 557-570 (1999).</li><li>Gasser, S. M., Daum, G., Schatz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Import+of+proteins+into+mitochondria.+Energy-dependent+uptake+of+precursors+by+isolated+mitochondria.">Import of proteins into mitochondria. Energy-dependent uptake of precursors by isolated mitochondria.</a> Journal of Biological Chemistry. 257 (21), 13034-13041 (1982).</li><li>Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., Weisbeek, 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+role+of+the+transit+peptide+in+the+routing+of+precursors+toward+different+chloroplast+compartments.">The role of the transit peptide in the routing of precursors toward different chloroplast compartments.</a> Cell. 46 (3), 365-375 (1986).</li><li>Lee, D. W., 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=Arabidopsis+nuclear-encoded+plastid+transit+peptides+contain+multiple+sequence+subgroups+with+distinctive+chloroplast-targeting+sequence+motifs.">Arabidopsis nuclear-encoded plastid transit peptides contain multiple sequence subgroups with distinctive chloroplast-targeting sequence motifs.</a> Plant Cell. 20 (6), 1603-1622 (2008).</li><li>Lee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+targeting+of+the+Arabidopsis+F1-ATPase+gamma-subunit+via+multiple+compensatory+and+synergistic+presequence+motifs.">Mitochondrial targeting of the Arabidopsis F1-ATPase gamma-subunit via multiple compensatory and synergistic presequence motifs.</a> Plant Cell. 24 (12), 5037-5057 (2012).</li><li>Jin, J. 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=A+new+dynamin-like+protein,+ADL6,+is+involved+in+trafficking+from+the+trans-Golgi+network+to+the+central+vacuole+in+Arabidopsis.">A new dynamin-like protein, ADL6, is involved in trafficking from the trans-Golgi network to the central vacuole in Arabidopsis.</a> Plant Cell. 13 (7), 1511-1526 (2001).</li><li>Lee, K. H., Kim, D. H., Lee, S. W., Kim, Z. H., Hwang, I. <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+import+experiments+in+protoplasts+reveal+the+importance+of+the+overall+context+but+not+specific+amino+acid+residues+of+the+transit+peptide+during+import+into+chloroplasts.">In vivo import experiments in protoplasts reveal the importance of the overall context but not specific amino acid residues of the transit peptide during import into chloroplasts.</a> Molecules and Cells. 14 (3), 388-397 (2002).</li><li>Lee, D. W., Lee, S., Oh, Y. J., Hwang, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sequence+motifs+in+the+rubisco+small+subunit+transit+peptide+independently+contribute+to+Toc159-dependent+import+of+proteins+into+chloroplasts.">Multiple sequence motifs in the rubisco small subunit transit peptide independently contribute to Toc159-dependent import of proteins into chloroplasts.</a> Plant Physiology. 151 (1), 129-141 (2009).</li><li>Lee, D. W., Woo, S., Geem, K. R., Hwang, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+motifs+in+transit+peptides+act+as+independent+functional+units+and+can+be+transferred+to+new+sequence+contexts.">Sequence motifs in transit peptides act as independent functional units and can be transferred to new sequence contexts.</a> Plant Physiology. 169 (1), 471-484 (2015).</li><li>Lee, 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=Both+the+hydrophobicity+and+a+positively+charged+region+flanking+the+C-terminal+region+of+the+transmembrane+domain+of+signal-anchored+proteins+play+critical+roles+in+determining+their+targeting+specificity+to+the+endoplasmic+reticulum+or+endosymbiotic+organelles+in+Arabidopsis+cells.">Both the hydrophobicity and a positively charged region flanking the C-terminal region of the transmembrane domain of signal-anchored proteins play critical roles in determining their targeting specificity to the endoplasmic reticulum or endosymbiotic organelles in Arabidopsis cells.</a> Plant Cell. 23 (4), 1588-1607 (2011).</li><li>Cleary, S. P., 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=Isolated+plant+mitochondria+import+chloroplast+precursor+proteins+in+vitro+with+the+same+efficiency+as+chloroplasts.">Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts.</a> Journal of Biological Chemistry. 277 (7), 5562-5569 (2002).</li></ol>

我们提供了一个详细的方案, 用于使用拟南芥原生质体研究蛋白质进口到叶绿体。该方法对研究蛋白质导入过程具有很强的功能。这种简单、通用的技术可用于检查预定货物蛋白的靶向叶绿体。利用该方法, 从拟南芥11、12的叶片组织中提取原生质体, 可在从非常早期到完全成熟的叶片的许多不同生长阶段提取原生质体。然而, 在种植用于原?...

叶绿体是植物中最重要的细胞器之一。叶绿体的主要功能之一是进行光合作用1。叶绿体还为脂肪酸、氨基酸、核苷酸和许多次生代谢物1, 2 的产生进行许多其他生化反应。对于所有这些反应, 叶绿体需要大量不同类型的蛋白质。然而, 叶绿体基因组只包含大约100个基因3,4。因此, 叶绿体必须从细胞溶胶中导入大部分蛋白质。事实上, 大多数叶绿体蛋白被证明是在翻译4,5,6后从细胞溶胶中导入的。植物细胞需要特定的机制来从细胞溶胶导入蛋白质到叶绿体。然而, 尽管这些蛋白质进口机制在过去几十年里一直在研究, 但我们仍然没有在分子层面上完全理解它们。在这里, 我们提供了一个详细的方法来制备原生质体和外生表达基因在原生质体。该方法可用于阐明蛋白质导入叶绿体的分子机制。
蛋白质的导入可以用许多不同的方法来研究。其中一种方法涉及使用体外蛋白质导入系统7,8。利用该方法,体外转化蛋白前体体体培养纯化的叶绿体, 用 sds-page 对蛋白质导入进行分析, 然后进行西方印迹分析。这种方法的优点是可以详细研究蛋白质导入叶绿体的每一步。因此, 该方法已被广泛用于定义蛋白质导入分子机械的成分, 并对过境肽的序列信息进行剖析。最近, 开发了另一种涉及使用叶组织原生质体的方法, 并已被广泛用于研究蛋白质导入叶绿体9,10.这种方法的优点是原生质体提供了一个比体外系统更接近完整细胞的细胞环境。因此, 原生质体系统使我们能够解决这一过程的许多其他方面, 如相关的细胞质事件, 以及如何确定靶向信号的特异性。在这里, 我们提出了一个详细的协议, 用于使用原生质体研究蛋白质的进口到叶绿体。

<table>
	<tbody>
		<tr>
			<td>GAMBORG B5 MEDIUM INCLUDING VITAMINS</td>
			<td>Duchefa Biochemie</td>
			<td>G0210.0050</td>
			<td></td>
		</tr>
		<tr>
			<td>SUCROSE</td>
			<td>Duchefa Biochemie</td>
			<td>S0809.5000</td>
			<td></td>
		</tr>
		<tr>
			<td>MES MONOHYDRATE</td>
			<td>Duchefa Biochemie</td>
			<td>M1503.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar, powder</td>
			<td>JUNSEI</td>
			<td>24440S1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore Surgical tape</td>
			<td>3M</td>
			<td>1530-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade stainless No.10</td>
			<td>FEATHER</td>
			<td>Unavailable</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Tube, 50ml</td>
			<td>SPL LIFE SCIENCES</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Macerozyme R-10</td>
			<td>YAKULT PHARMACEUTICAL IND.</td>
			<td>Unavailable</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulase ONOZUKA R-10</td>
			<td>YAKULT PHARMACEUTICAL IND.</td>
			<td>Unavailable</td>
			<td></td>
		</tr>
		<tr>
			<td>ALBUMIN, BOVINE (BSA)</td>
			<td>VWR</td>
			<td>0332-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>SIGMA</td>
			<td>M1902-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>CALCIUM CHLORIDE, DIHYDRATE</td>
			<td>MP BIOMEDICALS</td>
			<td>0219463505-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Twister</td>
			<td>VISION SCIENTIFIC</td>
			<td>VS-96TW</td>
			<td></td>
		</tr>
		<tr>
			<td>Screen cup for CD-1</td>
			<td>SIGMA</td>
			<td>S1145</td>
			<td></td>
		</tr>
		<tr>
			<td>Screens for CD-1</td>
			<td>SIGMA</td>
			<td>S3895</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>SPL LIFE SCIENCES</td>
			<td>10090</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>HILGENBERG</td>
			<td>3150102</td>
			<td></td>
		</tr>
		<tr>
			<td>LABORATORY CENTRIFUGE / BENCH-TOP</td>
			<td>VISION SCIENTIFIC</td>
			<td>VS-5500N</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>JUNSEI</td>
			<td>19015S0350</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>SIGMA</td>
			<td>P3911-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-GLUCOSE, ANHYDROUS</td>
			<td>BIO BASIC</td>
			<td>GB0219</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>DUKSAN</td>
			<td>40</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium nitrate tetrahydrate</td>
			<td>SIGMA</td>
			<td>C2786-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol)</td>
			<td>SIGMA</td>
			<td>P2139-2KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>SIGMA</td>
			<td>M2393-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube 13ml, 100x16mm, PP</td>
			<td>SARSTEDT</td>
			<td>55.515</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>MARIENFELD</td>
			<td>1000412</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses</td>
			<td>MARIENFELD</td>
			<td>101030</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting Chamber</td>
			<td>MARIENFELD</td>
			<td>650030</td>
			<td></td>
		</tr>
		<tr>
			<td>Axioplan 2 Imaging Microscope</td>
			<td>Carl Zeiss</td>
			<td>Unavailable</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tube 1.5ml</td>
			<td>SARSTEDT</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>SIGMA</td>
			<td>M3148-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS), Proteomics Grade</td>
			<td>VWR</td>
			<td>M107-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS, Ultra Pure Grade</td>
			<td>VWR</td>
			<td>0497-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT (DL-Dithiothreitol), Biotechnology Grade</td>
			<td>VWR</td>
			<td>0281-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue sodium salt ACS</td>
			<td>VWR</td>
			<td>0312-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>JUNSEI</td>
			<td>27210S0350</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Colors A.v. Monoclonal Antibody (JL-8)</td>
			<td>TAKARA</td>
			<td>632381</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying protein import into chloroplasts using protoplasts

叶绿体是一个重要的细胞器, 负责植物的各种细胞过程, 如光合作用和许多次生代谢物和脂类的产生。叶绿体需要大量的蛋白质来处理这些不同的生理过程。95% 以上的叶绿体蛋白是核编码的, 在细胞核糖体上翻译后从细胞溶胶中导入叶绿体。因此, 将这些核编码的叶绿体蛋白适当导入或靶向叶绿体对于叶绿体和植物细胞的正常运作至关重要。核编码叶绿体蛋白包含特定于叶绿体的信号序列。分子机械定位到叶绿体或细胞溶胶识别这些信号并进行导入过程。为了研究蛋白质在体内导入或靶向叶绿体的机制, 我们开发了一种快速、高效的原生质体基方法来分析蛋白质导入拟南芥叶绿体的情况。在这种方法中, 我们使用从拟南芥叶片组织中分离出的原生质体。在这里, 我们提供了一个详细的协议, 用于使用原生质体来研究蛋白质导入叶绿体的机制。

蛋白质导入叶绿体可以通过两种方法进行检测: 荧光显微镜和 sds-page 介导的分离后的免疫印迹分析。在这里, 我们使用 rbcs-nt:gfp, 一种融合结构编码 79 n-端氨基酸残基 rbcs 含有与 gfp 融合的过境肽。当蛋白质导入叶绿体时, 目标蛋白 rbcs-nt:gfp 应与叶绿素自动荧光的红色荧光信号合并, 如荧光显微镜检查 (图 1)。这两个信号的紧密重叠表明蛋白质导入叶绿...

Watch this Scientific Journal Video about Studying Protein Import into Chloroplasts Using Protoplasts at JoVE.com

Studying Protein Import into Chloroplasts Using Protoplasts

在这里, 我们描述了一种协议, 以表达蛋白质原生质体使用 peg 介导的转化方法。该方法为感兴趣的蛋白质的表达提供了方便的方法, 有效地研究了蛋白质定位和各种实验条件在体内的导入过程。

原生质体在叶绿体中导入蛋白质的研究

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many ...

这个谜团可以帮助回答细胞生物学领域的几个问题，如蛋白质输入叶绿体。该技术的主要优点是，在体内条件下可以研究叶绿体的蛋白质输入。演示程序将是李俊浩和杨菊康，两个后出我的实验室。首先，从一到两个B5板从两个星期大的植物中收获完整的叶组织。使用手术刀收获叶子，并把它们放入含有25毫升酶溶液的50毫升圆锥管中。将管子水平放在旋转摇床上，在黑暗中22摄氏度的温和搅拌下孵育8至12小时，直到溶液中只保留小毛花和茎。完成孵育后，通过140微米孔径网将含有释放的表层酶溶液过滤成新鲜的培养皿。然后在 50 毫升锥形管中小心地将溶液分层在 15 毫升 21%蔗糖溶液上。在98 gs下将管在摆动桶转子中离心10分钟，加速和减速设置最低。之后，使用帕图拉移液器小心地从最上层去除含有酶溶液的蛋白酶，以及酶溶液和蔗糖溶液之间的界面。将溶液转移到含有 30 毫升 W5 溶液的 50 毫升锥形管中，然后反转管进行混合。在51克时将管子离心6分钟。使用移液器小心丢弃此上流液，而不干扰颗粒中的原体。加入 25 毫升 W5 溶液，轻轻重新悬浮原压。为了稳定，在四摄氏度的冰箱中孵育至少一小时。经过4摄氏度的孵育，在46克左右离心两分钟，完全将原型丸粒育成。小心地完全去除超高动量，并将 MANG 溶液添加到原丸中，达到每毫升五倍十至六。使用移液器将 10 微克的血浆 DNA 和 300 微升的 protoplast 溶液添加到新鲜 13 毫升圆形底部管中。用手轻轻旋转管子，然后立即加入300微升新鲜准备的40%PEG溶液。通过几乎水平倾斜管子，用手轻轻旋转几次，完全混合。在室温下孵育30分钟。然后添加一毫升W5溶液，并像以前一样用手轻轻旋转管子完全混合。在室温下孵育样品10分钟。重复两次，先加入1.5毫升，然后加入两毫升W5，最后加入混合后，孵育30分钟。在46克时将管离心4分钟，然后丢弃上一代。将两毫升 W5 溶液加入颗粒中，然后以与以前相同的方式轻轻混合，但完全混合。在22摄氏度的黑暗室内孵育18小时。要使用荧光显微镜分析蛋白质导入，请使用带修剪尖端的移液器，在玻璃幻灯片上放置 10 微升的 protoplast 溶液。使用盖滑器小心地盖住溶液，以避免损坏前托帕斯特。将幻灯片放在荧光显微镜的舞台上，使用用于观察绿色荧光蛋白和叶绿素自荧光的激发入场过滤器。使用冷却充电两者设备摄像头捕获图像，并使用想象编辑软件处理图像以生成伪彩色图像。对于总蛋白质提取和免疫印迹，从原蛋白溶液中去除一毫升，然后混合剩余的一毫升。将这种原状溶液转移到离心管中，以 46 gs 的离心机进行 4 分钟。完成离心后去除上流液，并添加80微升变性缓冲液。漩涡活力五秒钟。添加五个 XDS 样品缓冲液，混合好，煮沸 10 分钟，变性。继续使用标准 STS 页面和具有抗 GFP 抗体的免疫印迹。RbcS 和 TGFT 是编码 79 端端氨基酸残留物的融合结构，含有与 GFP 融合的过境肽，用于研究通过荧光显微镜和免疫膨胀将蛋白质导入叶绿体。当通过荧光显微镜检查时，目标蛋白的绿色荧光信号与来自叶绿素自体浮游的红色荧光信号合并，表明蛋白质导入叶绿体。在隔离总蛋白后，在免疫布洛特中观察到两个蛋白带，表明一种蛋白质被正确导入叶绿体。上带对应于导入叶绿体后加工的全长前体和下带。蛋白质的加工形式以时间依赖的方式增加，表明蛋白质被导入叶绿体。在这一开发中，这项技术为细胞生物学领域的研究人员探索阿拉比多普斯的蛋白质累人或膜螺纹采摘铺平了道路。

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

mRNA Interactome Capture from Plant Protoplasts

mRNA从植物原生质体捕获

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

利用网格和收集荧光蛋白陶瓷的结构解

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological ...

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 ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

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 ...